E.R. Adlard Fd.), Chromatography in the Petroleum Industry Journal of ChromatographyLibrary Series, Vol. 56 0 1995 Elsevier Science B.V.

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CHAPTER 12

HPLC and column liquid chromatography

A.C. Neal
Esso Research Centre, Milton Hill, Abingdon, Oxfordshire OX13 6AE, UK

12.1 INTRODUCTION

The development of high performance liquid chromatography (HPLC) was predicted in 1941 by Martin and Synge [l]. In addition to pioneering liquidliquid chromatography and the theoretical plate model of chromatography, these authors predicted that HPLC would be achieved by using very small particles and a high pressure difference across the column. In fact, the origins of such columns can be traced back to the work of Tswett in 1903 [2] and their use was further extended by Kuhn and Lederer in 193 1 [3]. The advance of gas chromatography (GC) in the petroleum industry in the 1950s was such that liquid chromatography was effectively overlooked throughout that decade. This rapid exploration and application of GC rekindled interest in liquid chromatography as a complementary technique which could open up regions of solute polarity, molecular weight and bulk separation alien to GC. Commercialization of HPLC columns, pumps and detectors during the 1960s and early 1970s simplified operation of the technique and allowed potential users to apply it with relative ease. During this time, various terms were used to describe the new technique: modern liquid chromatography, high pressure liquid chromatography and high performance liquid chromatography and the latter is now universally used although it is not easy to define exactly what is meant by high performance. A comprehensive introduction to HPLC can be found in Snyder and Kirkland [4] although this excellent book is now almost 20 years old. More recently, Parris [5] and Gilbert [6] have written good general reviews on the subject and the reader is referred to reference [4] for detailed and fundamental information and the latter two books for general theory and instrumental descriptions.
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The applications of HPLC in petroleum analysis has itself been reviewed by Amos in 1979 [7]. At that time, the number of specific applications in the industry were few and the major uses were in hydrocarbon type analysis and the determination of hindered phenol antioxidants in fuels, most notably aviation turbine fuel. By comparison with paper chromatography and thin-layer chromatography, which had independently competed with HPLC during the earlier years of development, Amos concluded that the choice between PC, TLC and HPLC is now fairly clear cut and that HPLC should be used for all routine high-speed quantitative analysis. Since that review was written, HPLC pump, column and detector designs have advanced and expanded markedly, such that a far wider field of applications now exists. HPLC has also diversified into aqueous/ionic systems (ion chromatography) and high performance size exclusion chromatography (gel permeation chromatography), and been hyphenated with spectrometry including inductively coupled plasma emission spectroscopy (ICPES), nuclear magnetic resonance spectroscopy (NMR), Fourier transform infra-red spectroscopy (FTIR) and most importantly mass spectrometry (MS). The numerous attempts to interface HPLC with MS have resulted in a variety of LC-MS systems with each interface type having its own specific limitations and applications.
12.2 APPARATUS

A typical HPLC system (Fig. 12.1) is still composed of a pump, sample injector, column, detector and data recorder much as described by Amos [7]. However, considerable improvement and development of each component has taken place. Advances in columns and detectors have resulted in a wider range of separations and detection strategies being available.
12.2.1 Solvent reservoirs

Solvent reservoirs consist of purpose built glass bottles with a helium inlet and filter (for degassing) and a solvent outlet composed of a fritted particulate filter and PTFE outlet tube. Some systems even allow for a slight helium overpressurization of the reservoir to assist pump priming and prevent cavitation in the solvent inlet tubing.
12.2.2 Pumps

Pumps have progressed from single isocratic systems delivering premixed solvent, to purpose built binary, tertiary and even quaternary mixing systems

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Solvent Reservoirs

Detectors
Fig. 12.1.Elements of an HPLC system.

Data System

which premix the solvents to the desired composition and deliver them at the required flow rate, compensating automatically for pressure and viscosity effects which may occur during the mixing process. During the early 1980s, much debate took place over the relative merits of high pressure mixing (after the pump outlet) and low pressure mixing (at the pump inlet). High pressure mixing suffers from a number of drawbacks: chief among these is 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 solvents is present as less than 5% of the total. Low pressure mixing requires only one pump with the solvents proportioned and mixed before the pump head. Control by microprocessor or computer data systems allows for almost any shape of gradient (and flow) profile to be delivered. For these reasons, low pressure mixing, under either of the remote control systems given above, has come to dominate the market for LC gradient systems but some caution is still necessary in use. Firstly, for complete mixing, some systems rely on a fairly large volume mixing chamber on the outlet side of the pump. In certain applications, such as backflushing, a sharp gradient profile is desirable and this may be compromised by the hold-up volume of the chamber. In other words, if it is necessary to make sudden step changes in the gradient, the step may actually be a slope. Low volume ( 1 0 ~ 1 dynamic ) mixers, such as the
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LEE micromixer, are of considerable use in eliminating this problem. Secondly, efficient degassing of the solvent used to be a prerequisite for accurate mixing in order to eliminate cavitation in the mixing system and the detector noise and inaccurate flow rates that could result. In practice, improvement in the design of the solvent reservoirs and pump head geometry have reduced the occurrence of these problems, provided that the manufacturers advice is heeded. Gradient elution may also limit the choice of detector to be used, especially if the detector depends on changes in a physical property of the mobile phase itself. This issue is discussed in detail in the section on detectors. Three main types of pump, reciprocating piston, syringe and diaphragm, were all applied in the early days but the reciprocating piston pump now dominates the standard HPLC market where flow rates are at , or above, 1 ml min-*. Syringe pumps are best suited to lower flow rates and as such find more use in microbore HPLC where flow rates are typically well below 1 ml min-l. Reciprocating piston pumps operate by means of a rotating eccentric cam which drives a piston. The piston draws solvent into a cylinder through the inlet check valve during the return stroke. During the delivery stroke, solvent is expelled through the outlet check valve and hence ultimately to the column. These pumps are relatively inexpensive, simple to maintain, and deliver a constant flow of solvent over a wide range of flow rates. The piston drive is usually controlled by solid state pulsing circuits and a stepper motor. This allows for rapid refilling of the cylinder followed by swift repressurization of the solvent in the pump head and then a smooth, constant volume delivery until the end of the delivery stroke. The design of the cam and its eccentricity determine the smoothness of the flow profile. This is now so well defined that accurate, rapid refilling has become commonplace and methods for smoothing the profile such as large volume 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 the pump, invisible inside the box.
12.2.3 Sample injectors

Sample injectors are almost exclusively of the six-port valve type, although on-column syringe injectors were initially used. Injection valves are connected between the pump and the column and as close to the top of the column as practically possible. An interchangeable sample loop of discreet volume is connected to 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 that the loop is connected into the flowing mobile phase and the sample is thereby injected onto the column.

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Various designs of injectors exist, depending on the volume of sample available and the physical scale of the HPLC system. Internal microlitre-sized loop injectors are available for microbore applications, whereas for standard and preparative work, interchangeable sample loops up to 5 ml in volume can be used with standard Rheodyne or Valco valves. Loops are filled by a standard glass syringe with a Luer fitting, with or without a flat-ended needle according to the design of the sample inlet on the valve. Remote actuation of injection valves or flow switching valves is usually accomplished pneumatically or electrically. Typically the solvent delivery system or data station allows for timed events, one of which is the activation of the injection valve. In pneumatic actuation, the valve is turned by a supply of high pressure gas, usually air, in a purpose made pistodcylinder type actuator. The gas supply is itself delivered to the actuator by a solenoid valve. The solenoid valve opens or closes under the control of the solvent delivery system or data station, in order to pressurize or depressurize the actuator and hence operate the injector. Electrical actuators tend to be more expensive but are faster than pneumatic actuators. They use a synchronous, high torque electric motor, directly controlled by a relay closure or TTL switch. The valve may be coupled directly to the motor, minimizing the number of moving parts. Although faster and potentially more reliable than the pneumatic actuator, the extra capital cost is often the sole factor which mitigates against their use. In our experience, pneumatic actuators seldom give any cause for concern and the extra cost of electrical actuators is rarely justified.
12.2.4 Columns

Selection of the appropriate column is, of course, entirely dependent on the particular separation desired. Over the last 15 years, the technology of column design and manufacture has advanced markedly, as has the range and reliability of packings available. In the late 1970s, columns were almost exclusively 250 mm long 316 stainless steel with an internal diameter of 4.6 mm. End fittings were of solid 3 16 stainless steel and packings typically amorphous silica or alumina, or else silica with an octadecyl bonded phase, commonly referred to as ODS or C18. At that time, intermediate polarity stationary phases were beginning to excite interest but only amino (-NH,) and nitrile ( X N ) phases were readily available. Since then the range of HPLC applications has broadened considerably and advances in column chemistry and design have been fundamental to that progress. HPLC analysis can be placed in one of four categories largely by virtue of the column type used.
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Chapter I2

(1) Reversed phase: where the phase is of spherical silica with a non-polar hydrocarbon chemically bonded onto the surface or less commonly styrene-divinylbenzene beads. The mobile phase is polar and is most often a mixture of methanol and water, acetonitrile and water or tetrahydrofuran and water. One important variant of reversed-phase HPLC is reversed phase ion-pair chromatography (RP-IPC) where the analyte is ionizable or protonizable, and the mobile phase consists of a buffered aqueous mixture containing a counter ion of opposite charge to the analyte. (2) Normal phase: where the column is packed with spherical silica or with silica with a polar phase chemically bonded to it. Typical bonded phases include amino (-NH,) and nitrile (-CN) already referred to (and which may also be used in reversed phase mode) and phenyl, nitro or diol. Some specific phases such as dinitroanilinopropyl are also finding considerable use. The mobile phase is non-polar, typically heptane with or without the addition of small amounts of more polar solvents such as methylene chloride or ethyl acetate. (3) Ion exchange: consisting of sulphonate or quaternary ammonium functional groups chemically bonded onto silica or styrene/divinylbenzene polymer particles. Weak cations or anions can be separated without the use of buffer solutions as mobile phase, whereas strong cations or anions will require them. (4) Size exclusion or gel permeation: where solutes are separated by virtue of their size in solution. This technique has many petroleum applications for the determination of the molecular weight of polymeric lubricant additives but is not considered in detail in this chapter.
The range of columns currently available is, therefore, extremely wide, such that the separation of hydrocarbons, functional groups, ionic compounds, polymers and even enantiomers can be achieved. Column design has advanced from conventional columns to include disposable cartridges, radially compressed columns, metal-free columns made from polyetheretherketone (PEEK), and columns with adjustable end fittings which recompress the packing if voids develop, prolonging column lifetime. Most stationary phases are also available in microbore columns with internal diameters of 1-2 mm, which offer the advantages of reduced mobile phase consumption and greater mass sensitivity. By contrast, preparative scale columns packed with any of the aforementioned stationary phases (with the exception of diol) are also available off the shelf. These have internal diameters (i.d.) of 9 or 21 mm and can be used to recover larger quantities of analytes, either for further purification or for identification. A petroleum HPLC laboratory can serve a diverse group of needs including the analysis of fuels, lubricants, additives, waste water and refinery process

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samples. 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 be precise, sensitive and stable. In addition, HPLC detectors should have a large linear dynamic range, be insensitive to temperature and eluent composition, exhibit low noise and drift, and be simple and easy to maintain. Since the early days of HPLC, no single detector has been able to fulfil all these criteria as the flame ionization detector (FID) has done so admirably for GC. Instead, a range of detectors has evolved, based either on changes in the bulk properties of the mobile phase, or upon a selective property of the analyte(s). The subject has been frequently reviewed and descriptions of the main detector types can be found in any general HPLC text [5,6]. The reader is directed to Scott for a more detailed and mathematical treatment [81. The treatment here will be restricted to a brief discussion of those detectors which have found application in the petroleum industry. Even with this proviso, the majority of detector types currently available are still included.
12.2.6 Selective property detectors 12.2.6.I W-visible spectrophotometers

Ultraviolet detectors have been used since the early years of HPLC and remain the workhorse detector in the majority of laboratories today. Early examples were effectively converted spectrophotometers with the only modifications being associated with inclusion of a flow cell rather than a cuvette holder. These instruments were therefore based on prism diffraction or grating interferometry, A of interest could be selected in order to achieve maxisuch that the specific , mum solute sensitivity. The quantitation principle is the Beer-Lambert Law which states that the amount of UV or visible light absorbed will be directly proportional to the solute concentration. Sensitivity and limits of detection will therefore vary from solute to solute as a function of the individual compounds extinction coefficient. In extreme cases, where no UV or visible light chromophore is present in the solute, no absorption will take place and such solutes will not be detectable. This is the chief limitation of UV detectors and it is especially apparent in petroleum analysis because saturated hydrocarbons have no chromophore. A second major limitation is that mobile phases which themselves absorb
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Chapter 12

UV light effectively impose a wavelength cutoff on the system. Below this wavelength the absorption of the mobile phase itself is so strong that solutes , in the same region cannot be detected. with A The principle of detection within prism or grating instruments relies on the transmitted light of the chosen wavelength being cast onto a photomultiplier. Only one wavelength is observed that is often a compromise between sensitivity and selectivity.

I2.2.6.2Diode array detectors (DAD)

Diode array detectors (Fig. 12.2) effectively allow a much broader wavelength range to be acquired simultaneously, such that an entire W spectrum (more typically 200400 nm) can be captured repeatedly throughout the analysis. These detectors became commercially available in the early 1980s, have rapidly established themselves as reliable and sensitive, and have allowed ever more complex detection strategies to be employed. Of course the solute still needs a chromophore and the mobile phase W cutoff must be observed, especially in gradient elution. The principle of detection within the DAD relies upon an array of photodiodes of typically 0.5 nm resolution, such that the transmitted light after the flow cell is dispersed by a holographic polychromator and directed onto the linear photodiode array. Thus, by recording the signal output from one photodiode, the eluent is monitored at a single wavelength and by recording the output from all
Photodiode Array

gg/gg'
ElliosoidalMirror
I

Ellipsoidal Mirror

Deuterium Lamp

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

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the diodes, an entire spectrum is obtained. The major disadvantage of this system is that light of all wavelengths is present in the sample cell simultaneously and as such, fluorescent light may well be present at the wavelengths being monitored. In practice this is such an infrequent occurrence as to be of little consequence but it must always be borne in mind, especially if the mobile phase or solutes can be excited. The DAD can be used as a simultaneous multiwavelength detector to maximize sensitivity to each solute in turn, or to record entire spectra in order to examine peak purity, or to produce three-dimensional maps of wavelength versus absorbance versus time. All three modes offer the user more accurate quantitation than would be possible with a single wavelength dispersive W spectrophotometer. For research use, the ability to record an entire W spectrum of all the unknowns in a sample can give an early indication of solute identity. Coupled with retention behaviour, this can yield hypothetical structural information, or solute functionality, or carbon number, depending on the LC mode employed (normal or reversed phase).
12.2.6.3 Fluorescence detectors

UV light can interact with some solutes by exciting delocalized electrons into higher energy states above the normal ground state. When these electrons relax back to the ground state, the solute will emit most of the absorbed energy as light at a longer wavelength than that which excited it. In solutes where this decay is instantaneous or where it ceases immediately upon removal of the incident light, the solute is said to be fluorescent. It is possible to monitor the emission wavelength and filter out the excitation wavelength altogether, and this produces very high sensitivity, some two to three orders of magnitude greater than W absorbance and is therefore a highly desirable method of detection. In order to take advantage of the phenomenon, non-fluorescent compounds may be derivatized prior to analysis with a reagent to produce a fluorescent derivative. In petroleum analysis, fluorescence detection is most useful when the solute itself is highly conjugated and fluoresces naturally, as do many polynuclear aromatic hydrocarbons. Fluorescent light emerges from the sample at random angles and most instruments monitor the light emitted at right angles to the excitation beam. Some solvents have the ability to quench fluorescence such that the process is effectively suppressed. In particular very polar or aqueous mobile phases and buffered or ionic eluents are not recommended due to this phenomenon.
12.2.6.4Electrochemical detectors

Compounds which are electrically oxidizable or reducible can be detected electrochemically.In coulometric detectors the solute is completely electrolysed, whereas in amperometric detectors, the solute is only partially electrolysed.
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Chapter 12

Amperometric operation is more suited to flowing systems and is the commonest mode of electrochemical detection. In amperometric detectors, the solute concentration is directly proportional to the diffusion rate of the solute across the boundary layer to the electrode surface. The electrode current is therefore dependent not only on the solute concentration but also on its diffusion coefficient. A detailed treatment of electrochemical detectors can be found in Scott [8]. Electrochemical detectors rely on the mobile phase being electrically conductive and the most direct method of assuring this is to use buffer solutions. 12.2.6.5Flame 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 the FID compatible with HPLC in order to take advantage of its properties of sensitivity, known response and linear dynamic range. A number of mechanical transport systems have been developed originating with James in 1964 [9]. A moving wire was employed to carry the column effluent through a heated zone where the mobile phase was evaporated off, and then to another zone heated to a higher temperature in order to evaporate/pyrolyse the solutes and carry them into the FID in a stream of nitrogen. The chief disadvantages of transport detectors all lie with the transport mechanism itself. The wire, chain or disk has proved to be difficult to coat uniformly, different solvents evaporate at different rates, accumulation of remaining traces of solute give memory effects. These factors all contribute to relatively poor signal to noise ratios. Since only a small proportion of the solute is evaporated and detected, the sensitivity and large linear range of the F D are not utilized. In conclusion the compromises inherent in transport FIDs have meant that this detector is not widely used and its early promise for HPLC use remains largely unfulfilled. 12.2.6.6 Mass spectrometers The interfacing of mass spectrometers to GC instruments (GC-MS) is possible because both techniques are readily compatible. GC-MS is now one of the most powerful diagnostic tools available to analytical chemists. Interfacing HPLC to mass spectrometers (LC-MS) is much more difficult and has 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 it was not until the work of Homing et al. [lo], Scott et al. [l 13, and Arpino et al. [ I 21 in the 1970s that LC-MS was effectively achieved. Overcoming the relative incompatibility of the liquid phase eluent and the high vacuum required in the source of the MS has proved to be a severe chal-

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lenge. In addition, the higher molecular weight, lower volatility and chemical polarity of many compounds separated by LC make them less easily ionized than the 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 relevant ionization techniques such as fast atom bombardment (FAB) and atmospheric pressure chemical ionization (APCI) have been applied in order to surmount this problem. The mere modification of LC to make it more compatible with existing high vacuum, electron impact MS has not on its own proved sufficient, and the development of more compatible MS ionization and inlet systems has been necessary for the two techniques to merge successfully. The whole subject has been well reviewed recently by Niessen and van der Greef [13]. These authors list 26 distinct types of interfaces for LC-MS developed since 1972. The reader should consult reference [131 in respect of thermospray LC-MS and the particle beam interface, both of which have been successful in petroleum and 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 as mobile phases absorb in the most useful regions of the IR spectrum. Secondly, using absorption wavelengths away from solvent absorption bands has invariably resulted in less sensitivity and higher background levels. The exceptions to this have been where the analyte contains a carbonyl (C=O) group and in size exclusion chromatography of polymers. The former case is able to take advantage of the high extinction coefficient and hence high sensitivity of the carbonyl group. The latter application is able to overcome both low sensitivity and high background by virtue of the relatively high sample concentration required by SEC. Many of the limitations have been overcome or greatly reduced by Fourier transform infrared (FTIR) instruments. Modern FTIR spectrometers have signal to noise ratios over 100 times larger than energy dispersive instruments and as a consequence sensitivity is greatly improved. Their chief disadvantage is that of high cost and another disadvantage is incompatibility with reversed phase eluents. The combination of water absorption and band broadening due to hydrogen bonding conspire to reduce sensitivity and to limit the usable part of the IR spectrum. Proton nuclear magnetic resonance spectroscopy ('H NMR) has also been used as an on-line HPLC detector. This technique exploits the odd spin of the hydrogen nucleus, lH, in order to gain information on the environment of various hydrogen atoms in the analyte molecules. In this way, the signals due to methyl, methylene and aromatic protons in various molecular environments can be separated and quantified. Once normalized, the proportion of various hydrogen types
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can be calculated and the alkyl, aryl and heteroatom substituents present in a sample elucidated. Proton NMR will be unable to distinguish hydrogen atoms from the mobile phase from those of the analyte and these will be included erroneously in the normalization if present. For this reason, static NMR experiments or LC-NMR cannot use standard solvents but are required to use perhalogenated or perdeuterated solvents. This is a severe limitation to on-line LC-NMR since these solvents are extremely expensive, especially if significant volumes of perdeuterated solvents such as chloroform-d (where 99.8% of the hydrogen is replaced by deuterium) have to be used for the LC separation. Another considerable limitation is the high capital and running cost of a modern Fourier transform NMR spectrometer. Nevertheless, this technique has found application in petroleum analysis and is expected to find increasing use.
12.2.7 Bulk property detectors

12.2.7.1 Refvactive index detector

The refractive index detector remains the second most widely used LC detector after the UV detector. It is universal, detecting all analytes whose refractive index (RI) differs from that of the mobile phase. The RI of a substance is a dimensionless constant which typically decreases with increasing temperature. Three types of RI detector are available and all are I betermed differential refractometers, that is they measure the difference in R tween a sample cell and a reference cell containing mobile phase only. It follows, therefore, that all refractometers are sensitive to temperature changes and to changes in eluent composition. Thus , in order to use them for gradient elution, the reference cell must always contain a mobile phase of identical composition to that in the sample cell and this is often impossible to achieve. For good baseline 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 Sample on one side, reference eluent on the other. Light from the source passes through this cell to a mirror behind it and is reflected back through the cell to a photomultiplier. If a solute of different RI enters the sample cell, the light beam will be deflected. The photomultiplier output is proportional to the magnitude of the deflection. Deflection RI detectors are simple and have a wide linear dynamic range. Instruments manufactured by Waters Associates have typically been of this design. Fresnel refractometers pass parallel incident light through a prism onto sample and reference simultaneously. If the refractive index of the liquid in the sample cell differs from that in the reference cell, some light from the sample

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cell will be diffracted, reducing the intensity of the beam reflected back out of the sample cell. The difference between the intensities of the sample and reference beams is measured by a photomultiplier and recorded. The linear concentration range of this detector is less than that of the deflection instrument unless two separate prisms are used to cover the entire RI range. The optical cleanliness of the system is also more critical than for the deflection detector. Fresnel refractometers have been manufactured by Perkin Elmer. Interference refractometers split the source beam, pass it through sample and reference cells simultaneously and then recombine it. Any difference in refractive index between sample and reference cells will manifest itself as a difference in optical path length when measured by an interferometer. This design is more sensitive than the previous types and additional sensitivity is possible if a laser is used as the light source as by Woodruff and Yeung [14,151. In summary, RI detectors are universal and can be sensitive under carefully controlled conditions. Their use in gradient elution is still far from straightforward and base line drift is to be expected when the mobile phase composition changes even by relatively small amounts. Despite all these operational drawbacks, they are still the detector of choice when the solutes have no UV chromophore, especially in isocratic determinations of saturated hydrocarbons.

12.2.7.2Evaporative light scattering detectors

The evaporative light scattering detector (Fig. 12.3), evaporative analyzer or mass 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 of operation, construction and performance of what is now referred to as the mass detector can be found in that reference. In essence, this type of detector consists of a nebulizer, evaporation chamber, light source, scattering chamber and light trap and a photomultiplier set at 135 to the incident light beam. Column eluate is nebulized with a relatively high flow of nitrogen or air and the mobile phase evaporated as the solvenugas mixture passes down the vertically mounted evaporation chamber. At the bottom of the chamber, all that is left is gas, solvent vapour and finely divided droplets or particles of analyte. This aerosol passes through the light beam and the photomultiplier 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 effectively independent of the RI of the analyte. The true linear working range of this instrument is not extensive, typically 1.5 orders of magnitude in concentration. Above and below this range, the size of the analyte droplets produced no longer promote the reflection and refraction of the light. Although this is a drawback, it is a relatively minor one, as the reReferences pp. 3 72-3 74

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Chapter 12

Nebuliser aa

118

II I

=Exhaust

Fig. 12.3. Schematic of an evaporative m a s detector.

sponse functions due to Rayleigh and Mie scattering in the non-linear regions are well described and still make calibration possible. The chief disadvantage of this detector is the volatility limitations imposed upon the analyte. The solvent evaporation chamber is, in effect a mild blowdown apparatus which removes the mobile phase. If the analyte volatility or vapour pressure approaches that of the mobile phase it will vaporize and give no response. In our laboratory, we have found hexadecane (b.p. 256C) to be partially evaporated when the detector is operating at ambient temperature with hexane (b.p. 68C). It is therefore likely that hydrocarbons below n-CI7will not give full recovery. Even given this limitation, the detector finds considerable use for intermediate and low volatility analytes.
12.2.7.3 Dielectric constant detector

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

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A typical DCD is a differential, temperature controlled concentric cylindrical capacitor through which the column eluent flows. The cell electrodes are made of stainless steel and are connected electrically as one side of a Wein or Schering Bridge. If the mobile phase is less polar than the analyte, as in normal phase LC, the dielectric constant of the eluate will increase as the peak elutes. The reverse is usually the case in Rp-HPLC, and in order to avoid negative peaks in reversed phase applications, DCDs allow for polarity reversal. Setting up and balancing DCDs can be a tedious business as each side of the bridge circuit needs to be balanced in an iterative fashion until the potential difference across the bridge is zero. The linear dynamic range of the DCD is heavily influenced by the difference in the dielectric constants of the mobile phase and the analyte, but has been quoted as 3.5 X lo4 which is comparable to the RI detector.
12.3 QUANTITATION

In the majority of petroleum applications of HPLC, calibration is by external standardization and quantitation is by peak area. Where samples are analyzed as received or after dilution only, this approach is reliable and accurate. Where the sample is worked up before analysis by liquidliquid or liquidsolid extraction, it is necessary to determine the extraction efficiency (or recovery) in order to be certain that a representative extract has been obtained. Where extraction efficiencies are low or where time does not allow the recovery to be determined, an internal standard or a standard addition method should be employed, provided the detector response to the solutes is linear in the range of interest. Peak area is most usually used for quantitation, as this is the most statistically precise measure of analyte concentration. It does presuppose good resolution however, and where this is not the case, a range of deconvolution methods or even peak height measurement may have to be considered. Contemporary HPLC now has a vast range of competitive quantitation devices and statisticaVgraphica1 software available. Stand alone benchtop integrators, microprocessor and PC data stations, local area networks (LANs), laboratory information management systems (LIMS) and even mainframe chromatography packages are all available. Selection is a compromise between cost, specification and, increasingly, compatibility with existing computer hardware. Any of these devices can take detector output and convert it to a high quality graphical or numerical report, automatically labelled with peak identities according to previously recorded retention windows. Caution is necessary, however, as any system will only act according to the way it is configured by the operator. At each stage of the data systems applicaReferences pp. 372-374

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tion, the user must be certain that each setting is sound in order to obtain a final quantitative output of the highest possible integrity.
12.4 APPLICATIONS

The applications of HPLC in petroleum analysis are summarized in Table 12.1. The wide variety of separation mechanisms, column chemistries and detection systems represented by HPLC offers the petroleum chemist a range of distinct 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, saturates, olefins and aromatics in petroleum products; (3) preparative or semi-preparative fractionation of complex mixtures for determination by other analytical techniques.
Within each category, standard methods exist for particular determinations, which have been rigorously tested in terms of inter-laboratory precision. Such standard methods as exist within the Institute of Petroleum handbook, Standard f Analysis and Testing o f Petroleum and Related Products, 1993 [19] Methods o are discussed in the following sections.
12.4.1 Individual compounds
12.4.1.1 Polycyclic aromatic hydrocarbons (PAHs)

These compounds have attracted considerable interest due to their role as pollutants and, in some cases, their carcinogenic properties. Amos [7] cites some early W L C applications. Katz and Ogan [20] have used partition and size exclusion columns in series to effect the analysis, and a combination of normal phase amino and reversed phase C 18 columns has been used to determine PAHs in crude oil by Grimalt and Albaigks [21]. Further LC-LC methods, chiefly aimed at benz[a]pyrene, have been employed by Tomkins and Griest [22] and Fielden and Packham [23]. In the former case, Partisil silica and analytical scale Vydac 201TP reversed phase columns were used and in the latter case cyclodextrin and ODS silica. In both cases, the selectivity and sensitivity of fluorescence detection was used to determine the PAH directly. Symons and Crick [24] have determined PAHs in refinery effluent after cleanup and preconcentration using a Radial-Pak CIS column with 75:25 acetonitrilelwater eluent and UV and fluorescence detection. Recoveries were vari-

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TABLE 1 2 . 1 APPLICATIONS OF HPLC IN PETROLEUM ANALYSIS Crude oil ha-arenes Dibenzothiophene Phenols Polynuclear aromatic hydrocarbons (PAHs) Preparation of PAH fractions Saturates/aromatic types Naphthdgasoline Aromatic nitrogen compounds Benz[a]pyrene Saturateshromatic types Grimmer et al. Rebbert et al. Christensen and White MacCrehan and Brown-Thomas Grimalt and Albaiges Ostman and Colmsjo Welch and Hoffman Nondek and Chvalovsky Tomkins and Griest Apffel and McNair Cookson et al. Munari et al. Hayes and Anderson ASTM D 2002,2003 ASTM D 1319 Nondek and Chvalovsky IP 374 Fielden and Packham IP PM-AT Welch and Hoffman Cookson et al. Hayes and Anderson Haw et al. Davies et al. ASTM D1319 Hayes and Hillman IP 343 Schabron and Fuller Nondek and Chvalovsky IP 391,PM-AY Lienne et al. Fielden and Packham Davies et al. Marshman IP PM-AZ Apffel and McNair Cookson et al.

Saturates fractions Saturates/olefindaromatics Aviation fuel Aromatic nitrogen compounds Coumarin PAHS Saturateshrornatics Saturateshromatic types

Saturates/olefindaromatics 2,4-Dimethyl-6-tertiarybutylphenol DieseUdistillatefuels Alkyl nitrates Aromatic nitrogen compounds Mono/di/triaromatics Olefins PAHS Phenalenones Saturatedaromatics Saturatedaromatic types

References pp. 3 72-3 74

364
TABLE 12.1 (continued) Davies et al. Hazlett el a1

Chapter 12

Diesel exhaust particulates Naphtho[8,1,2-abc]coronene

Nitrated PAHs

Jinno et al. Paputa-Peck et al. MacCrehan e f al. Tomkins and Griest Musha et al. Musha et al. ASTM D 2549 Saito DeSanzo et al. Mazzeo et al. Palmentier et al. Ostman and Colmsjo DeSimone et al. IP 368 ASTM D2007 Pei et al. Pei and Hsu Bear ASTM D3712 Chen and Nero Blanco-Gomis et al. Fodor and Newman

Fuel oil Benz[a]pyrene Lubricating oils Additives (over 50)

Aromatichon-aromatic fractions Renz[a]pyrene

Furfural Naphthalene/phenanthrene PAH fractions

Polychlorinated biphenyls Saturates/aromatics Saturatedaromaticsipolars Sulphonates

Sulphurized alkylphenols V1 improver Zinc dialkyldithiophosphates

Heavy oils Olefinic fractions PAH fractions Saturates/aromatics/PAH/resins/asphaltenes, etc. Saturates/naphthenes/alkylaromatics/thiophenes

Bitumen PAH fractions

Yamamoto and Akutsu Coulombe and Sawatzky Lancas et al. Hsu et al. Hsu et al.
Coulombe and Sawatzky Symons and Crick

Refinery effluent PAHs

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able and less than 87% for 4-6 ring PAHs. Saito [25] determined benz[a]pyrene in lubricating oils and greases with fluorescence detection after an alumina clean-up; precision was reported as 6 7 % RSD with a detection limit of 3.95 nglg. HPLC has also been used purely as a fractionation technique for PAHs. Coulombe and Sawatzky [26] applied this method to bitumens and heavy oils and determined PAHs in the various LC fractions by GC. Palmentier et a l . [27] employed a semi-preparative scale fractionation followed by GC-MS. Ostman and Colmsjo [28] prepared PAH fractions from crude oil and used crankcase oil by elution from short silica columns followed by an automated backflushed Bondapak-NH, HPLC system. Individual PAHs in the final fractions were quantified by GC. Detection limits were in the order of 1 ppm from a 10-15 mg sample using GC-FID or 0.1 ppm by scaling up the initial silica clean-up. l . [29] detected PAHs as quinones by oxidizing them with CeN. Mazzeo et-a Reductive mode electrochemical detection was employed to achieve detection limits in the order of ppb. Chromatography was performed on an ODS column using propan-2-01 /phosphate buffer as eluent. These authors applied the above system to the analysis of naphthalene and phenanthrene in a motor oil. A proposed Institute of Petroleum standard method, IP PM-BN, also exists for the determination of PAHs in petroleum, coal and shale oil products. Detection limits of 0.1% d m of total, and 0.1 mg kg-1 of individual PAHs are quoted. The method uses open, gravity feed silica columns to produce a PAH extract which is further separated by HPLC on a 5,um particle Spherisorb amino column or equivalent. The isolated 4-6 ring fraction is then run on a Sephadex LH20 partition column in order to separate alkylated PAHs from the parent PAHs. These parent PAHs are individually determined by GC. Precision has yet to be established.
12.4.1.2 Other indigenous compounds

Nitrated PAHs in diesel engine exhaust particulates have been examined by l . [30] and MacCrehan et al. [31]. Paputa-Peck employed a Paputa-Peck et a normal phase HPLC fractionation of methylene chloride extracts. Determination of individual nitrated PAHs was by GC with a nitrogen-phosphorus detector or by GC-MS. MacCrehan separated methylene chloride extracts by RP-HPLC and compared voltammetry, amperometry and fluorescence for direct detection of individual compounds. Diesel particulates have also been examined by Jinno et a l . [32] for naphth0[8,1,2-abc]coronene using reversed phase separations and multichannel W detection. High molecular weight heterocyclic nitrogen and sulphur compounds have l . [33] and Andreolini et al. [34]. These authors used been studied by Borra et a
References pp. 3 72-3 74

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highly efficient capillary LC columns and a combination of direct diode array fluorescence detection or fraction collection and mass spectrometry to examine solvent refined coal distillate, syncrude and shale oil samples. Polycyclic aromatic nitrogen compounds (aza-arenes) in Arabian Light crude oil have been examined and identified by Grimmer et al. [35]. These authors used a TLC/HPLC isolation scheme with separation and identification of individual compounds by GC. Aza-arenes, anilines and alkyl aromatic amines in gasoline, kerosene and diesel fuel have also been studied by Nondek and Chvalovsky [36] using two different charge transfer columns, 3-(2,4dinitrobenzene su1phonamido)propyl silica and 3-(2,4-dinitroanilino)propyl silica. A comparison of five different charge transfer columns for the separation of aromatic compound classes from a crude oil distillate sample and other fossil fuel samples has been made by Thompson and Reynolds [37]. Phenalenones such as 7H-benz[d,e]anthranen-7-one, benzanthrone and methyl phenalones have been quantified in middle distillates by Marshman [38] using silica reversed phase separation and UV detection at 400 nm. Detection limits quoted are typically in the region of 0.2 mg I-*. Dibenzothiophene has been quantified in crude oils by Rebbert et al. [39] and by Christensen and White [40]. The former authors employed HPLC to fractionate samples for GC/FPD quantification. In contrast, the latter authors used a novel LC-tandem MS system to separate and unambiguously identify dibenzothiophene directly. Indigenous phenols in crude oil have been examined by MacCrehan and Brown-Thomas [41] with detection limits of less than 100 ng/g. These authors used alkaline solvent extraction of the oil, solid phase purification of the extract and RP-HPLC with electrochemical detection. HPLC has even been applied to asphaltenes in order to assist the determination of an average molecule. Monin and Pelet [42] used size exclusion and a range of bonded phase columns to fractionate such samples after selective dissolution in a number of solvents.
12.4.I . 3 Additives and contaminants

The anti-oxidant 2,4-dimethyl-6-tert-butyl phenol has been quantified by normal phase isocratic HPLC with UV detection and is the subject of an IP Standard Method, IP343. The method allows a number of columdmobile phase combinations and, in our experience, is robust and precise. Published repeatability and reproducibility at the 200mg/l level are 2.61 and 6.56, respectively. Some homologues and isomers of this compound may also be separated using variations in mobile phase composition. The same compound has also been quantified with electrochemical detection by Hayes and Hillman [43]. Alkyl nitrate cetane improvers in diesel fuel have been determined by Schabron and Fuller [44]. Normal phase LC on silica coupled with variable

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wavelength IR detection was used to separate and quantify amyl, hexyl and octyl nitrates. Recovery, accuracy and precision quoted were good and detection limits of 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 determined by Musha et al. [45,46]. These authors used both normal and reversed phase columns with UV detection at the maximum absorbance for each compound. Furfural has been determined in lubricating oils by Di Sanzo et al. [47]. A 5 p m ODs-silica column with an eluent of 70:30 watedmethanol and W detection at 280 nm gave a recovery >95%, good precision, and good agreement with a bisulphite extractionKJV method. Samples for HPLC were pre-extracted with methanol and cleaned-up with a C18 silica cartridge prior to determination. Synthetic and indigenous sulphonates, including alkyl benzene sulphonates, have been separated and quantified by Bear [48]. This author evaluated the evaporative light scattering detector in the analysis of a wide range of surfactants and concluded ...a uniform linear response for each class of surfactant, with detection limits in the low nmole range. In particular, the detector response was reported to be independent of the alkyl chain length and the degree of aromaticity with respect to alkyl benzene and alkylaryl petroleum sulphonates. Columns and mobile phase varied according to the application and samples were analyzed after dilution of the parent product. A diode array UV detector was also used in series with the ELS detector. Standard deviations of all the analytes were less than 1%. Sulphurized alkylphenols have been separated from reaction side products and base oil on a normal phase, y-cyclodextrin silyl column with gradient elution and evaporative light scattering detection by Chen and Nero [49]. Individual fraction from the separation were also characterized by mass spectrometry. Fingerprint comparison between samples which passed and failed engine test specifications are presented. The advantages of the ELSD over RI detection were stated by these authors to be freedom from ambient temperature variation effects, minimal baseline drift with multiple solvent gradients and a response which was mass dependent rather than concentration dependent. To illustrate the breadth of HPLC applications in the field of lubricating oil additives, normal phase and reversed phase methods have even been applied to the characterization of poly(styrene-alkylmethacry1ate)co-polymer viscosity index improvers of molecular weight up to 300 000 Da by Blanco-Gomis et al. 1501. An Institute of Petroleum method exists for the determination of the coumarin content of kerosene. This compound, 1,2-benzopyroneis often added to kerosene as a marker for excise purposes. The method uses a silica column, a mobile phase of 2% propan-2-01 in hexane or heptane and W detection at 274nm.
References pp. 3 72-3 74

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Typical calibration range is 2-4 mg/l and at the 2 mg/l level, repeatability is quoted as 0.06 and reproducibility 0.28.
12.4.1.4 Compound classes

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

HPLC and column liquid chromatography

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column to separate alkene-free gasolines into saturates, monoaromatics, diaromatics, tri/polyaromatics and polar groups. Each group was quantified by an RI detector into weight percentage abundance. Calibration samples were obtained by fractionation of a fuel sample rather than by use of single pure compounds in an effort to minimize compositional and RI response factor differences. Both Apffel and McNair and Munari et al. used on-line HPLC-GCRID methods to analyse gasoline saturates, unsaturates, aromatics and polar compounds. The latter authors employed a retention gap between the two chromatographic systems and microbore HPLC columns. Hayes and Anderson used off-line HPLC with a dielectric constant detector to achieve an accurate group type separation and quantitation of gasoline with uniform response factors from the detector. The mobile phase was 2,2-dichloro-1, 1,1-trifluoroethane (Envron 123). The individual fractions were then analyzed by GCMSD to identify components and GC/FID to quantify them. The authors reported that spent Envron 123 can be reused several times without purification or easily redistilled on a continuous basis. Kerosenes have been characterized also by some of the authors previously cited [51,53,55]. In addition, Haw et al. [56] used a propylamino silica column with on-line NMR as the detector. Ln this case, the mobile phase was l,l,ltrichlorotrifluoroethane with 2.5% deuterochloroform and 0.05% hexamethyldisiloxane as NMR reference. Each compound class (monocyclic and dicyclic aromatics) could be given an average composition. The average composition of the saturate fraction was, however, limited by problems in accounting for quaternary carbon. Davies et al. [57] utilized the LC-retention gap-GCRID approach to a kerosene sample with microbore amino and silica glass-lined LC columns in series with pentane eluent and backflushing. Unfortunately, the low dead volume of detectors required for microbore LC precluded conventional RI or dielectric constant detectors and thus direct quantitation of the saturate and aromatic fractions prior to GC was not possible. The system was automatic and clearly improved the analysis of the aromatic fractions. Diesel and distillate fuels have been studied by all the methods described for crudes, gasolines and kerosenes [52,53,57]. Silica and amino columns have been used to separate diesel into saturates, olefins and aromatics with RI and/or U V quantitation by Felix et al. [58]. Davies et al. [59] used the LC-GC technique previously described, but with specific reference to polynuclear aromatics in diesel fuel. The chromatographic system described by these authors produced a complete fractionation by compound class but in this study emphasis was placed on the definition of a two-dimensional (LC versus GC) retention map for selected PAHs. By comparison, the retention indices of aromatic compounds from the diesel sample led the authors to conclude that naphthalene, phenanthrene and their alkyl derivatives were the predominant aromatics present.
Refeences pp. 3 72-3 74

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The LC-NMR approach, previously applied to kerosene, has been extended to diesels and distillates by Hazlett et al. [60], again culminating in the determination of average composition for each compound class. From LC-NMR data, Caswell et al. and Swann found it possible to predict the physical properties of diesel and jet fuels [61,62]. Multiple regression analysis was used for the correlation of 13 LC-NMR parameters from each fuel with 17 physical properties such as cetane number, distillation data, flash point, pour point, density, etc. Thirteen of the 17 properties in reference [61] had correlation coefficients in excess of 90% and seven were in excess of 95%. Copper(li) and silver-modified silica columns have been prepared by passing ammoniacal CuSO, through the column or by use of ammoniacal AgN03 during packing by Lienne et al. [63]. With pentane or Fluorinert FC72 as mobile phase, I and UV deolefins could be separated from light and heavy distillates with R tection. Heavy hydrocarbons have been characterized by Hsu et al. [64,65] by on-line LC-MS. It was reported that distinction could be made between naphthenoaromatics and alkylaromatics and also between aromatic hydrocarbons and thiophenes. The value of this kind of information for refinery processing is very high.
12.5 PREPARATIVE HPLC AND COLUMN LIQUID CHROMATOGRAPHY 12.5.1 Standard methods

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

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ASTM D2002 and D2003 are also silica gel fractionation methods used to provide representative saturate fractions from low and high olefinic naphthas, respectively. Again 2-propanol is used under pressure as the mobile phase. ASTM D2001 uses a dual column system of Attapulgus clay and clay/silica gel. Saturates, aromatics and polar fractions from oils with initial boiling points greater than 260C are recovered from the columns. This analysis is often referred to as the Clay-Gel method. Once again it is a time-consuming and labour intensive technique. This type of separation has been the subject of HPLC development by Pei et al. and by Pei and Hsu [67,68]. ASTM D2549 furnishes aromatics and non-aromatics for further analysis by mass spectrometry. In this method, up to 10 g of oil boiling between 232 and 538C are separated on a column of bauxite and silica gel. Pentane is used to elute the non-aromatics and the aromatics are eluted with successive portions of diethyl ether, chloroform and ethanol. ASTM D3712 uses a silica gel column with chloroform and ethanol to separate diluent oil from petroleum and synthetic sulphonates. The average molecular weight of the sulphonate is then determined by ashing the recovered material. Metal sulphonates are first hydrolysed to sulphonic acids and converted to sodium sulphonates prior to the separation. Incompletely hydrolysed samples do not separate well and, as the analysis is again labour intensive, care must be taken in the hydrolysis step to avoid a considerable waste of effort. This method is identical with IP Method 369.
12.6 INDIVIDUAL PUBLICATIONS

Zinc dialkyl dithiophosphate additives (ZDDPs) in finished lubricating oils have been determined on 183 cm X 0.78 cm glass columns packed with 3775pm silica by Fodor and Newman [69]. The alkyl/aryl ratio of the ZDDPs in the recovered methylene chloride fraction was determined by IR spectroscopy against calibration solutions of the pure compounds. The authors concluded that losses incurred in the chromatography limited the method to that of an estimation rather than a quantitative determination. Yamamoto and Akutsu used a preparative scale silica gel column to separate saturates and aromatics and a 60 A silica/lO-20% w/v silver nitrate column to separate saturates from alkenes. The argentation column could produce a 2 g fraction of alkenes from a column packed with 80 g of argentated silica. Typical samples included heavy distillates from thermal cracking [70]. De Simone et al. [71] obtained polychlorinated biphenyl extracts from petroleum samples by means of a combination of gel permeation and silica microcolumns. The PCB concentration in the extract was determined by GC with electron capture followed by mass spectrometry to confirm structures.
References pp. 372-374

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A mixed heavy end sample was separated into fractions with a 50 x 1 1 cm silica 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 mixtures used to fractionate it. Six solvents in six mixtures of increasing eluotropic strength gave saturates, monoaromatics, diaromatics, triaromatics, polynuclear aromatics, resins, asphaltenes and asphaltols. Typical recovery is quoted as better than 90%in most cases, with an RSD of 1.2%.
12.7 FUTURE TRENDS

A number of more selective column mechanisms are beginning to find application in petroleum analysis. Most specifically, the range of selectivities now commercially available in normal phase charge transfer columns such as DNAP, TNAO and TNAP columns are allowing a more precise definition of aromatic type cut point. As the industry has a continuing need for more precise total aromatic and aromatic type quantification, it is expected that the use of such columns will increase. Similarly, the separation of functionalized bad actors from a range of hydrocarbon products may prove to be accomplished by anion and cation exchange columns which are now also commercially available. Undoubtedly, the single most useful advance in detector design for the petroleum industry has been that of the evaporative mass detector. This detector will find increasing use in the field for two reasons. Firstly, the operation of the detector necessarily results in volatile sample matrices being evaporated along with the HPLC mobile phases used in petroleum applications. This may actually prove to be an advantage in the analysis of some gasoline additives. Secondly, as heavier products will not suffer the same fate as gasoline/naphthas, characterization of such samples can take advantage of the detectors true linearity and composition independence. These characteristics are unique in such a robust and relatively inexpensive device. Finally, LC-GC is still waiting for an enterprising manufacturer to develop a truly turnkey system. Numerous applications of this hyphenated technique already exist which should be transferable. The analysis of oxygenates in gasoline at percentage and at trace levels may yet prove to be the application which arouses a sufficient volume of interest to be commercially viable.
12.8 REFERENCES
1 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. 6. 3 R. Kuhn and E. Lederer, Ber. Deut. Chem. Ges., 64 (193 1) 306.

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4 L.R. Snyder and J.J. Kirkland, Introduction to Modem Liquid Chromatography, Wiley, New York (1974). 5 N.A. Parris, Instrumental Liquid Chromatography, Elsevier, Amsterdam (1984). 6 M.T. Gilbert, High PerformanceLiquid Chromatography, Wright, Bristol(l987). 7 R. Amos, in Chromatography in Petroleum Analysis (Altgelt and Gouw, Eds.), Marcel Dekker, New York. (1979). 8 R.P. W. Scott, Liquid Chromatography Detectors, Elsevier, Amsterdam (1977). 9 A.T. James, J.R. Ravenhill and R.P.W. Scott, Chem. Inst., 746 (1974). 10 E.C. Homing, D.I. Carroll, I. Dzidic, K.D. Haegele, M.G. Homing and R.N. Stillwell, J. Chromatogr. 99 (1974) 13. 11 R.P.W. Scott, C.G. Scott, M. Munroe and J. Hess Jr., J. Chromatogr., 99 (1974) 395. 12 P. Arpino, B.G. Dawkins and F.W. McLafferty, J. Chromatogr. Sci., 12 (1974) 574. 13 W.M.A. Niessen and J. van der Greef, Liquid ChromatographyMass Spectrometry, Marcel Dekker, New York (1992). 14 S.D. Woodruff and E.S. Yeung, Anal. Chem., 54 (1982) 1174. 15 S.D. Woodruff and E.S. Yeung, Anal. Chem., 54 (1982) 2124. 16 D.L. Ford and W. Kennard, J. Oil Colour Chem. Assoc., 49 (1966) 299. 17 D.L. Ford and W. Kennard, Aust. Pat. Appl. No. 33406. 18 J.M. Charlesworth, Anal. Chem., 50 (1978) 1414. 19 Standard Methods of Analysis and Testing of Petroleum and Related Products, 1993, Institute of Petroleum and Wiley, London. 20 E. Katz and K. Ogan, 5th Int. Symp. on Chem. Anal. Biol. Fate Polynucl. Aromat. Hydrocarbons, Battelle Press, Columbus, OH (1980) pp. 169-178. 21 J. Grimalt and J. Albaiges, Afinidad, 40(385) (1983) 223. 22 B.A. Tomkins and W.H. Griest, J. Chromatogr., 386 (1987) 103. 23 P.R. Fielden and A.J. Packham, J. Chromatogr., 479 (1989) 117. 24 R.K. Symons and I. Crick, Anal. Chim. Acta, 151 (1983) 237. 25 T. Saito, Bunseki Kagaku, 39 (1990) 21 1. 26 S. Coulombe and H. Sawatzky, Fuel, 65 (1986) 552. 27 J.P.F. Palmentier, A.J. Britten, G.M. Charbonneau and F.W. Karasek, J. Chromatogr., 469 (1989) 241. 28 C.E. Ostman and A.L. Colmsjo, Fuel, 68 (1989) 1248. 29 J.R. Mazzeo, I.S. Krull and P.T. Kissinger, J. Chromatogr., 550 (1991) 585. 30 M.C. Paputa-Peck, R.S. Marano, 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. 31 W.A. MacCrehan, W.E. May, S.D. Yang and B.A. Benner Jr., Anal. Chem., 60 (1988) 194. 32 K. Jinno, J.C. Fetzer and W.R. Biggs, Chromatographia, 21 (1986) 274. 33 C. Borra, D. Wiesler and M. Novotny, Anal. Chem., 59 (1987) 339. 34 F. Andreolini, C. Borra, D. Wiesler and M. Novotny, J. Chromatogr., 406 (1987) 375. 35 G. Grimmer, J. Jacob, K.W. Naujack and D. Schneider, Erdoel Kohle, Erdgas, Petrochem., 38(2) (1985) 82 (Synopsis 8504). 36 L. Nondek and V. Chvalovsky, J. Chromatogr., 3 12 (1984) 303. 37 J.S. Thompson and J.W. Reynolds, Anal. Chem. 56 (1984) 2434. 38 S.J. Marshman, Fuel, 5 (1990) 364. 39 R.E. Rebbert, S.N. Cheder, F.R. Guenther and R.M. Parris, J. Chromatogr., 284 (1984) 211. 40 R.G. Christensen and E.V. White, J. Chromatogr., 323 (1985) 33. 41 W.A. MacCrehan and J.M. Brown-Thomas, Anal. Chem., 59 (1987) 477.

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