A Practical Guide to HPLC Detection || OTHER MODES OF DETECTION

CHAPTER 9

OTHER MODES OF

DETECTION Colleen Parriott Sparta, N.J. 07871

I. INTRODUCTION

Many detection techniques other than those discussed in the previous chapters are used for HPLC. Some of these lesser used techniques will be discussed here, but no attempt has been made to make this chapter all-inclusive.

These techniques are usually used for one of two reasons. Either other techniques such as UV or refractive index cannot be used because the analyte is not responsive to the technique (i.e., doesn't absorb UV or differ enough from the solvent for refractive index) or because more information is required. In the latter case, the detector is usually used in series with UV or refractive index. All the detectors discussed in this chapter present problems that make them less popular than those discussed in the preceding chapters. These problems are usually solvent related. In some cases, such as evaporative light scattering, the solvent must be removed that requires the use of heat or nebulizers. In other cases such as flow-through IR, solvent remains and obscures part of the spectrum.

Solvent gradients cause problems in nearly all of these detection techniques, although it is much more serious in some cases than in others.

It should be noted that nearly all of these techniques could be used off-line by collecting fractions and analyzing them, but this is very labor intensive.

This chapter is divided into five sections. The first four cover a particular technique or class of techniques in detail. The last gives brief introductions to some other techniques.

A Practical Guide to HPLC Detection 233 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

234 Colleen Parriott

II. RADIOACTIVITY

A. Introduction

1. General The spontaneous emission of radiation from an atom resulting from a change

in its nucleus is a phenomenon called radioactivity. Using this phenomenon, individual atoms may be labeled without any significant change in their chemical properties and traced through metabolic pathways and reaction mechanisms. Radiochemical techniques are very sensitive, very selective and require a minimum of sample preparation.

Radioactivity is a random event. It is not possible to accurately predict when a given atom will disintegrate, but watched over time, radioactive decay takes the form of a first-order reaction. Statistically accurate data of the decay can be obtained if it is followed for a long enough period of time. Higher levels of activity require shorter periods of time than lower activity levels.

2. Half-life The time it takes for half of the atoms of a given radioactive substance to decay is called its half-life. This is a characteristic number for a given isotope: 5760 years for 14C, 12.26 years for 3H, and 8.04 days for 131I. [1]

3. Units Radioactivity is given in many units. Here, only three are of concern: counts per minute (CPM], disintegrations per minute (DPM), and curies.

DPMs are the number of atoms in a given sample that disintegrate in one minute. CPMs are the number of disintegrations registered by a detecting device in one minute. The terms disintegrations per second (DPS) and counts per second (CPS) are also sometimes used. CPMs are related to DPMs by the equation

CPM = DPM x efficiency of the counting device

Most on-line radioactivity detectors can give data in either CPM or DPM, providing the efficiency is known.

A curie is the quantity of a nuclide that disintegrates at a rate of 3.7 x 1010

atoms per second. As one would expect, a millicurie is one thousandth of a curie and a microcurie is one millionth of a curie. These units are used when buying and using radioactive materials. They are not normally associated with HPLC detectors.

4. Radioisotope Effects When dealing with radioactivity in general, and radio HPLC specifically, the assumption that a labeled molecule behaves the same as an unlabeled one should be verified if possible. This should be done for both the pathway being measured [2] and for the HPLC analysis.

Worth and Retallack [3] studied the isomer effects in the HPLC separation of tritium-labeled vitamin D metabolites. In all the systems they studied, the

9 · Other Modes of Detection 235

labeled derivatives behaved like more polar compounds than their unlabeled counterparts. They attributed the observed effect to hydrogen bonding differences between OH groups and OT (T is tritium) groups, and the difference in polarity between C-T bonds and C-H bonds.

Cundy and Crooks [4] studied the effect of unlabeled nicotine on the separation of 14C labeled nicotine enantiomers. The presence of the pure unlabeled enantiomer was found to cause the labeled racemic mixture to separate into two enantiomer peaks. They proposed that this is due to differential association of the two labeled enantiomers with the unlabeled standard.

5. Radioactivity Types Before proceeding to a discussion of radio HPLC detection, a short review of alpha, beta, and gamma radioactivity is given. For a more in-depth discussion of these activity types, Friedlander et cd. [5] is recommended. For a discussion of other radioactivity types see Moe et al. [6] and Greiner et al. [7].

Alpha radiation is the emission of a helium nucleus (2 protons, 2 neutrons) from an unstable atom. Alpha particles from a given isotope are monoenergetic or with only a few discrete energy levels. This type of radiation is usually found only in heavy nuclei.

Beta radiation is the emission of an electron or a positron, accompanied by a neutrino (with electron emission) or an antineutrino (with positron emission). Neutrinos and antineutrinos are particles of zero rest mass and no charge.

Whereas alpha radiation is emitted at discrete energy levels, beta radiation is seen as a continuous energy distribution. The sum of the energy of an electron and a neutrino or a positron and an antineutrino is constant, thus energy is conserved. Each isotope has a characteristic energy profile, which is utilized when two beta emitters are monitored simultaneously.

As an atom disintegrates by alpha or beta emission, the nucleus is often left in an excited state. Deexcitation may occur by the emission of high-energy electromagnetic radiation called gamma radiation. Gamma rays, like alpha rays, are emitted at discrete energy levels characteristic of a given isotope.

Isotopes that emit gamma radiation with beta emissions include 1311,22Na, 198Au and 150. Other nuclei such as 3H, 14C, 32P, and 35S do not emit gamma rays with their beta emissions and are therefore called pure beta emitters.

In HPLC detection, beta radiation is most often of interest, particularly for 14C and 3H. It is detected with scintillation counting and will be considered for the bulk of this section. Gamma radiation is also often of interest, particularly 131I. Its on-line detection is done by several techniques and will be discussed at the end of the radioactivity section. Alpha radiation is not of interest for online HPLC as often as other radiation types. When it is analyzed it is usually done with the same scintillation counting techniques used for beta radiation. It will not be specifically covered here except to cite work done by Zhu et al. [8]. They used a solid scintillant on-line to detect 241Am and 242Cm to obtain efficiencies of 85.8% and 92.8%, respectively. More recently, Bartholdi et al. demonstrated picogram detection levels for 238Pu [118].


B. Fraction Collection versus On-Line Detection

1. Comparison Once a mixture containing radioactive components is separated by liquid chro-matography, one must decide the mode of detection. Two methods frequently employed are fraction collection followed by scintillation counting and on-line (flow-through) scintillation counting.

Fraction collection is a labor intensive process. It involves collecting fractions, preferably very small ones, adding a scintillator, loading vials into a scintillation counter, and working up the data.

Flow-through or on-line detection involves hooking up a radioactivity detector at the end of the column and observing results.

Flow-through detection is obviously less labor intensive and faster. Also, because fractions do not have to be collected, smaller samples can be analyzed, better resolution is obtained and, in homogenous detection, less scintillation fluid is used [9]. Data analysis and integration is usually easier because detectors normally have computer data analysis systems. In cases where elution is used, flow-through detection is advantageous because differences in solvent viscosity will not affect the size of the fraction collected and analyzed [10].

So why not always use flow-through detection? The main limitation is the level of activity that can be accurately measured. In on-line detection counting time is very limited, and background levels tend to be about twice that seen in static detection [11]. Both of these limit the accuracy of counting samples of low radioactivity levels. As a general rule, samples giving peaks of at least 100 CPM are good candidates for on-line detection.

Fractions collected may be counted for as long as desired, so low activity levels can more accurately be analyzed. The actual detection limits of flow-through systems vary with the isotope measured and the scintillation system used.

Kessler [12,13,14] compared on-line detection with fraction collection of steroid metabolites. He found the mean ratio of DPM on-line/DPM fraction collection was 0.784, demonstrating that fraction collection is more sensitive. The on-line detector gave two major peaks and four or five minor ones, while the fraction collection method gave two major peaks and one minor one (see Fig. 9-1) [14]. This demonstrates the better resolution that is obtained with online detection. Comparable resolution could be obtained with fraction collection, but very short collection times would be required. For the above to work, fractions would have to be collected every six seconds for 40 minutes, yielding 400 vials to be counted.

2. Automated Fraction Collection Some methods have been used to automate counting in systems of lower radioactivity levels. Baba et al. [15,16] have designed a five-cell synchronized accumulating radioisotope detector. This is a system where eluate leaving the column is collected in the first cell, counted, and exited to a second cell. Meanwhile, the first cell is refilled with new eluate. The process is repeated for five cells and then a computer analysis of the total count yields a chromatogram. This


14.0

12.0

^ 10.0 CO

Ξ 8.0

X 6.0 CO

Έ Q. 4.0 O

2.0·

Ό

X w 0.5-I Έ Q_ Q

40

ΙΛΛ. 20 30 40

FIG. 9-1 The upper panel is a plot of CPM/aliquot of fractions from the Ridi Rac 2112 Fraction Collector. The lower panel is the actual plot of the Flo-One HP Radioactivity Detector signal for aH dpm. (Reprinted from Kessler, M. (1982) /. Liq. Chromatogr. 5(2) 313-325. Courtesy of Marcel Dekker Inc.)

has been successful for homogeneous systems; heterogeneous systems show excess peak broadening [15] and need more work.

A second system for automating fraction collection and analysis was demonstrated by Karmen et al. [17,18,19]· Their totally automated procedure starts with the collection and concentration of fractions. The concentrated solutions are transferred to filter paper [17] or silica gel TLC plates [18] and assayed by autoradiography on photographic film, followed by densitometry for quantification.

The use of a spacer liquid combined with a storage loop is a third procedure [20,21]. Here the eluate is combined with a suitable immiscible "spacer liquid" and sent to a storage loop. It stays there until the run is over. The direction of flow is then reversed and the sample in the spacer liquid is sent back to the detector to be counted for a suitable period of time. Bakay [22,23] used a similar procedure for a high-pressure amino acid analyzer. In all cases the spacer liquid served to prevent the eluate from spreading and causing peak broadening.

These techniques will not be discussed in more detail. The following discus-


sion will be limited to cases where radioactivity levels are high enough for online detection.

C. General Aspects of Beta Detection

1. Overview When a decay event takes place it is not directly measured. Instead, the column effluent is put in contact with a material that will become excited by emitted radioactivity and emit light at a detectable wavelength. This material is called a scintillator.

2. Homogeneous versus Heterogeneous Detectors for beta radiation fall into two general categories: homogeneous and heterogeneous. The difference between them is the scintillator type. In heterogeneous detection the detector contains a tube of scintillator beads or grains (solid scintillator) through which the column eluate passes. In homogeneous systems, the liquid column eluate is combined with a liquid scintillation mixture and run through the detector. An excellent detailed description of static homogeneous scintillation is given by Cooper [1].

As would be expected, detector properties vary with the scintillator and the isotope being analyzed. In general, homogeneous detectors give higher efficiencies, lower detection limits, and less background noise than heterogeneous detectors. Macek et al. [24] compared detection properties of 3H and 14C for both types of detectors. They obtained efficiencies of 50 and 90% for 3H and 14C, respectively, in the homogeneous mode, and 8.and 85%, respectively, for the hetergeneous mode. Typically, tritium gives lower efficiencies because it is weaker in energy. Detection limits, also given by Macek et al., were 100 and 200 DPM for 14C and 3H, respectively, for the homogeneous mode and 250 and 900 DPM, respectively, for the heterogeneous mode. Frey and Frey [25] reported similar results when they compared homogeneous and heterogeneous counting of different commercial detectors. They found dynamic efficiencies ranging from 26.7 to 31.4% for homogeneous counting and 4.2 to 17.2% for heterogeneous counting of 3H.

3. Detection Variables Three things ideally maximized in radiochemical detection are speed, resolution, and sensitivity. The three physical parameters of a detector varied to adjust these are flow rate, detector volume, and scintillator type. Scintillators are covered in Sections U.E.3 and II.F.3 and will not be discussed here.

a. Speed With other detector types, speed would not be considered a factor in detection, but radioactivity is a random event. The longer it is measured, the more statistically accurate the reading becomes. This is why samples with low activity levels are not good candidates for on-line detection.


b. Resolution Resolution is the ability to distinguish between two adjacent peaks. If resolution is inadequate, it can be improved by increasing peak separation or decreasing peak width.

c. Sensitivity Sensitivity is the ability to detect the radionuclide over background and other factors such as chemiluminescence. Minimum detectable activity is included with sensitivity. Also covered here is detector efficiency, which plays a role in determining sensitivity. (In this chapter, "efficiency" always refers to detector efficiency, and not to Chromatographie efficiency.)

Minimum detectable activity (MDA) is usually considered two [26], but may be considered up to ten times [27] the background level of radiation. Assuming the level is twice the background, the following equation can be used [26]:

MDA = B x W/[T x E)

where B is background in counts per minute, W is peak width in minutes, T is residence-time minutes (cell volume/flow rate), and E is efficiency.

Efficiency is the percent of radioactive events recorded by the detector. It may be calculated as [26]:

%E = Observed activity (CPM) x 100/Known activity (DPM)

The above equation is particularly well suited to static systems. For flow systems efficiency is more easily calculated by injecting a standard

of known activity in DPM and recording the total counts in the integrated peak. Percent efficiency is then calculated as

%E = S x F x 100/(Std. x V)

Where S is total number of counts in the sample peak, F is flow rate in milliliters per minute, V is volume seen by the photomultiplier tubes, and Std. is the known amount of injected radioactivity.

It was mentioned above that sensitivity is the ability to detect radionuclides over factors such as chemiluminescence. Chemiluminescence can prove to be a serious problem in some systems, where it is seen as high, sporadic background noise. It is best handled by removing compounds that cause the problem, such as peroxides in ether [25]. In addition some commercial detectors have compensation circuits to help supress luminescence [28,29]. Sensitivity, detection limit, and efficiency are dependant of the nuclide being detected.

4. Detector Variables

a. Detector volume It was stated above that detector volume and flow rate are two detector variables. As a flow cell becomes larger, sensitivity is increased, due to a larger sample size, and resolution is decreased due to peak broadening. Therefore detector volume is a tradeoff. Maximum resolution and maximum sensitivity cannot both be obtained; the objective of detection must be evaluated [30].

A flow cell should be chosen that has a volume one-half to one-fourth the volume of the smallest peak of interest. This is expressed in an equation as [26]


V = K x P for solid scintillator cells

V = K x (P + S) for liquid scintillator cells

where V is flow-cell volume, P is volume of the smallest peak in milliliters (peak width times flow rate), K is a constant between \ and \ where smaller values give greater resolution and larger values give greater sensitivity, and S is volume of scintillator fluid in milliliters for the same period as the peak.

b. Flow rate The second detector variable is flow rate. It, like detector volume, is a trade-off, here between speed and sensitivity [30]. As the flow rate is increased, sensitivity is decreased due to a shorter residence time in the detector, and speed is increased. A flow rate is normally chosen that will give adequate sensitivity in as little time as possible.

D. Basic Equipment Description

On-line radioactivity detectors were designed for automatic amino acid analyzers [31], and for column chromatography systems [32-38] before high-performance liquid chromatographs became readily available. These helped to lay the groundwork for the development of HPLC on-line detectors.

The main parts of an on-line detector are photomultiplier tubes, a flow cell, and a miocroprocessor. The detector is either interfaced directly to the HPLC column or follows in-line with another detector, such as UV.

The flow cell is a piece of tubing, usually Teflon, through which the sample passes. It is hollow for homogeneous detection and filled with solid scintillator grains for heterogeneous detection. The flow cell is usually designed in a spiral shape to give the sample as much analysis time as possible for good sensitivity [39], with as much surface exposure as possible for good resolution [40].

Flow cells are situated in the detector in a manner that prevents light penetration and may be shielded with lead to reduce background noise. On each side of the flow cell is a photomultiplier tube (PMT). The PMTs receive the light given off by the scintillator and convert it into easily measured electrical signals.

Two PMTs are used around a flow cell to lessen the problem of electrical noise or dark current. A pulse of light is received by both PMTs at the same time, but noise is random and usually would be registered by only one PMT at a time. Coincidence circuitry is used in which only pulses received simultaneously by both PMTs are registered [1,40].

The electrical signal from the PMTs is amplified and sent to a pulse-height analyzer. This is where data for individual isotopes is determined in dual nuclide samples. It may also be used to remove background noise [39] that is too high or too low in energy to likely have come from the measured isotope. These data are then sent to a computer for further analysis and printing.

In homogeneous detection, the detector will also have a pump for adding scintillation fluid and possibly an electronic stream splitter. The stream splitter is located before the pump. It combines a specified amount of the eluate with


scintillation fluid and sends it through the flow cell. The remaining eluate is sent to a fraction collector.

Figure 9-2 shows a block diagram of a radioactivity HPLC detector.

E. Homogeneous Beta Detection

1. Advantages Homogeneous detection is most useful for analytical applications where quanti-tation of relatively low levels of radioactivity is of prime importance. The main advantage of this type of detection is that it is more sensitive than heterogeneous detection: Efficiencies are higher and detection limits are lower, particularly for 3H and 14C. This was discussed in Section II.C.2.

Two other advantages are that no sizable back pressure develops and that radioactive materials seldom build up in the detector. The importance of these two properties is that they are the major disadvantages of heterogeneous detection.

2. Disadvantages and Considerations The major problem with homogeneous detection is that scintillation fluid is used. The first problem caused by scintillation fluid is that the fluid is not easily removed, therefore the sample is destroyed. If sample recovery is of importance, an electronic stream splitter can be used as was employed by Kessler [12,13,14] and Roberts et al. [10]. This separates the column effluent stream into two streams; one is combined with scintillator fluid and passed through the detector,

HPLC Column

Electronic Stream Spli t ter

Scinti l lation! Fluid

Reservoir Pump

Mixing Tee

Flow Cell

Fract ion Col lector

Coincidence Circuitry

Pulse Height Analyzer

Computer

Printer

To Waste

FIG. 9-2 Block diagram for an HPLC radioactivity detector.


the other is sent to a fraction collector. This reduces the resolution and sensitivity of the chromatogram, but Kessler [12] showed that the resolution reduction may not be substantial. The amount of material collected in the fraction collector is obviously less than would be obtained in heterogeneous detection.

Other problems caused by scintillator fluid include Cost The fluid must be purchased initially and disposed of

later. Waste disposal This is expensive and not environmentally preferable.

Equipment The detector must have a scintillator pump and its auxiliary equipment.

Toxicity Scintillator fluids are usually toxic. In using homogeneous detection two important issues must be kept in mind. First the column eluate and scintillator fluid must be well mixed to give the best detection [10,41]. A pulse dampener may be necessary, depending on the type of pump used [30].

The second issue is quenching. Recall that detection of beta radiation involves the transfer of energy from an emitted beta particle to a scintillator, which then gives off light at a detectable wavelength. If a compound or a solvent is present that stops the scintillator from absorbing the radiation energy or absorbs the light that the scintillator gives off, counting efficiency will drop.

Although in some cases quenching may originate from components in the sample, it normally originates in the solvent and only needs to be considered when an elution is used. When there is no gradient, any quenching caused by the solvent is constant throughout a run and is not a problem, unless a very strong quenching solvent is used. Quenching caused by the solvent gradient is neither constant nor linear [42] and therefore must be accounted for. Birkle et al. [2] assessed the effect of acetonitrile/water on counting efficiency and found a 2% change in efficiency over the gradient. The magnitude of quenching is solvent dependent. Some commercial detectors come with programs that can automatically correct for counting efficiency variations in acetonitrile/water and methanol/water systems [43]. For systems where quench data is not known, a plot of efficiency versus solvent composition for the range of interest should be prepared. This plot, called a quench curve, is then programmed into the detector and used to correct for elution.

Strong quenching agents include ketones, amines, and esters. For more on quenching see Cooper [1] and Reeve et al. [30].

3. Scintillation Fluids Numerous scintillation fluids are available from numerous vendors, many of which are specifically designed for on-line detectors and specific application types. On-line applications require that the fluid choosen be nongelling, as not to clog the system. For high counting efficiencies, the fluid must have a high holding capacity for, and be readily miscible with the eluate. This allows the detector to see a homogeneous solution, which is particularly important when water or salts are present. If the eluate has any quenching molecules, a fluid that has energy transfer agents such as naphthalene should be considered. Other aspects one may wish to consider are flash point, toxicity, cost, and applicability to specific experimental conditions (high or low temperature, etc.).


Another aspect to consider is how much scintillator fluid to use. That is, what ratio of scintillator fluid to column eluate should be used? Ideally as little fluid as possible to achieve good results is used. Extra fluid is expensive and causes increased waste. Commercial scintillation fluids usually recommend a range of ratios to consider. If a quenching agent is present, a higher ratio may be necessary.

Macek et al. [24] studied how scintillator flow rate affects peak area and detection efficiency in 14C and 3H detection. In their study, the flow rate of material through the column was held constant at 0.6 mL/min so that as the scintillator flow rate increased, so did the scintillator-to-eluate ratio. The detector volume was also constant so increased scintillator flow rate caused a decreased residence time. The peak area shows a maximum at a scintillator flow rate of about 2 mL/min (Fig. 9-3). At flow rates above this, the decrease in residence time caused a decrease in peak area. This caused the optimal scintillator-to-eluate ratio to be 3.33 (2 :0.6 mL/min).

The plot of efficiency versus scintillator flow rate (Fig. 9-4) shows a steady increase until about 2 mL/min, at which point it begins to level off. This is typical of systems in which some quenching is present. When no quenching is present, the maximum is reached and then the efficiency begins to drop off. Choosing the ratio of scintillation fluid to eluate should therefore consider quenching, sample activity level, and the type of fluid being used.

F. Heterogeneous Beta Detection

1. Advantages Solid scintillator detectors are most useful when purification of radioactive compounds is the main objective, and in cases where high-energy beta emitters are being studied. The advantages of this type of detection over liquid-liquid systems deal mainly with the absence of scintillator fluid.

3500

Έ

CO Φ

CO 0)

CL

2000

500

Flow rate (mL/min)

FIG. 9-3 Relationship between scintillator flow rate and peak area (D, 14C; Δ, 3H). (Reprinted from Macek, J., Lichy, A., Pesakova, V., Adam, M. (1989) /. Chromatogr. 488 267-274 (Courtesy of Elsevier Science Publishers.)


90%

Flow rate (mL/min)

FIG. 9-4 Relationship between detection efficiency and scintillator flow rate (D, 14C; Δ, 3H). (Reprinted from Macek, J., Lichy, A., Pesakova, V., Adam, M. (1989) /. Chromatogr. 488 267-274 (Courtesy of Elsevier Science Publishers.)

Products can be easily and totally recovered since there is no scintillation fluid contaminating them.

It is cheaper since no fluid needs to be purchased or disposed of. Equipment is simpler since no scintillator pump is necessary. It can be used before or after other detectors. Quench correction is simpler or not required.

2. Disadvantages The problems with heterogeneous detection include contamination of the beads, high back pressure, and low efficiencies.

Contamination of the scintillator grains is caused by materials such as lipids, proteins, peptides, and steroids, particularly over 6000 molecular weight, adhering to the grains. The radioactive material will then slowly bleed off, causing "memory effects" seen as higher background counts and peak broadening.

Memory effects are difficult to deal with. Several approaches have been tried to either prevent adherence or remove material already adhered. Methods to prevent adherence include silaniation of glass beads [44], which was unsuccessful; preincubating the beads with unmarked material [29], and combining the eluate with ammonia [24], both of which proved to be successful in some cases. Removal of adhered material has been attempted using ammonia [24], commercial detergent followed by acid [45], ozone [44], and surfactant [Brij, 35] [38], all of which may work in certain cases. Also tried were solutions of salts, solutions of dilute acids, and organic solvents, which were mostly ineffective [44]. Frequently the only solution for serious adsorption problems is replacement of the scintillator grains, or switching to homogeneous detection.

It is a good idea to check the entire HPLC system for memory effects from time to time. This is done by injecting solvent blanks and recording detector output. Memory effects will be seen as high background radiation.


High back pressure is a second problem frequently encountered. Back pressure is a particularly serious problem when pressure sensitive components such as detectors are used in sequence prior to the radioactivity detector.

The pressure built up in a heterogeneous detector depends on several factors.

ScintiJJator grain size As grain size is decreased, both efficiency and back pressure increase.

Flow rate As flow rate is increased, back pressure increases. Eluate viscosity Back pressure increases when viscosity is in

creased. Flow cell Length, width, and shape.

If a commercial detector is used, the flow cell is normally a flat coil and is not easily varied. If a standard method is being used, or materials of limited solubilities are involved, eluate viscosity may likewise be difficult to change. If the solvent system can be changed, acetonitrile/water mixtures are less viscous than methanol/water mixtures [46]. Flow rate may be lowered, but this is at the expense of speed, and may not be practical. Scintillator grain size is often the easiest to change, but this is at the expense of efficiency. As with memory effects, if back pressure cannot be controlled, homogeneous detection may be a better choice.

The third problem for solid scintillators is low efficiencies, particularly when low-energy beta emitters such as 3H are being studied. This was discussed in Section II.C.2. High-energy beta emitters usually show acceptable efficiencies in heterogeneous detection and are therefore prime candidates for this type of detection.

3. Solid Scintillators Solid scintillators are beads or grains of material that absorb radiation energy and emit visible light, which is readily detected by photomultiplier tubes. As the grain size is decreased, efficiencies increase, as was demonstrated by Mackey et al. [44] for several glass scintillators (Table 9-1). Unfortunately, memory

TABLE 9-1

Efficiency as a Function of Scintillator Type and Size (for

Scintillator size

38-63 ptm 63-90 μτη 90-125 μπι Unsieved

NE901

Efficiency (%)

73.0 ± 3.4 66.7 ± 2.2 63.0 ± 1.6

FMa

65

75

NE913

Efficiency (%)

62.3 ± 2.4 57.3 ± 2.3 44.0 ± 1.1 40.7 ± 1.5

FM

111 75 39 36

GS1

Efficiency (%)

71.8 ± 1.2

63.6 ± 1.4

glass

FM

99

75

scintillators)

KG3L

Efficiency (%) FM

51.9 ± 1.7 67

46.8 ± 1.9 48

Reprinted from Mackey, L., Rodriguez, P., and Schroeder, F. (1981). /. Chromatogr. 208, 1-8. Courtesy of Elsevier Science Publishers. a FM (figure of merit) = (efficiency)2/background


effects and back pressure also increase when grain size is decreased, which limits the size particle that may be used.

A solid scintillator must have several characteristics to be effective and practical. It must be able to withstand the pressures the system will put on it without changing shape, being crushed, or forming gaps. It must pack well and be available in the proper grain size. It also must be resistant to any solvents it may come in contact with [47].

The scintillator must also have an acceptable efficiency for the isotope being measured. Wunderly [48] studied the efficiency of several isotopes using cerium-activated yttrium silicate glass. His study, which included alpha, beta, and gamma emitters, demonstrated that (1) efficiencies varied greatly with the isotope, and (2) given proper conditions, heterogeneous efficiencies may surpass homogeneous efficiencies for some isotopes. The data also demonstrate that high-energy beta and gamma emitters give higher efficiencies than low-energy beta emitters.

Several types of solid scintillators have been used, as is shown in Table 9-2. Because efficiencies are dependent on several factors, such as grain size and flow-cell shape, wide ranges are sometimes reported.

Some of these compounds displayed acceptable efficiencies, but had limitations due to their physical properties. PPO is soluble in 0.05N HC1 and in many

TABLE 9-2

Sol id Scinti l lator Efficiencies

Solid scintillator

Anthracene Calcium fluoride,

Eu activated 2,5-Diphenyl-

oxazole (PPO) Lithium glass,

cerium activated (NE 901, NE 913)

2,2 '-p-phenylene-bis-(5-phenyloxazole) (POPOP)

2-(4 '-tert-butylphenyl-5-(4"-biphenyl-l,3,4-oxadiazole

Yttrium silicate, cerium impregnated

% Efficiency

3H

1-2 0.5-5

1-1.8

0-2

1.7

12

14 C

31-44 38

43

40-99

50-54

40

90

Reference

36, 48 36, 48, 40

36, 48

36, 44, 48

38

36

48

Comments

See Table 9-1

a

Other efficiencies: 1-125 75% P-32 95% Am-241 160%

a Th i s reference gives relative efficiencies for naphthalene; trans-stilbene; 4,4'-diphenylstilbene (DPS); 2-(naphthyl-(l'))-5-phenyloxazole (ANPO); p-terphenyl (PTP); 1,1,4,4-tetraphenylbutadiene-(1,3) (TPB); PPO; and NE901.


organic solvents such as toluene. In 50% ethanol it broke down and clogged the cell. It also had high memory effects and did not respond well to pressure. Anthracene and butyl PBD are likewise soluble in organic solvents and 50% ethanol. In addition they dissolve in 2N HCl. PTP reportedly requires very high pressure to maintain an acceptable flow rate [38].

Cerium-activated lithium glass does not dissolve in any common solvents except hydrofluoric acid and is frequently the scintillator of choice. Eu-activated calcium fluoride is also commonly used, but it is somewhat soluble in ammonium salt solutions and should be avoided in these cases. Yttrium glass is also frequently used.

G. Gamma Radiation

1. Properties In Section ILA.5 above, a brief introduction was given for gamma radiation. In addition to being very energetic, gamma radiation is very penetrating. This property makes it conducive to a wider variety of detection methods than was seen for beta radiation.

Before going into actual detection methods, a phenomenon of gamma radiation first theorized by Cerenkov in 1934 will be mentioned. Cerenkov radiation is "light emitted by a high-speed charged particle when the particle passes through a transparent, nonconducting solid material at a speed greater than the speed of light in that material" [49]. The material used for Cerenkov radiation detection depends on the energy levels of the isotope of interest. Gamma radiation is emitted at discrete energy levels characteristic of a given isotope.

2. Gamma Detection Gamma radiation may be detected on-line using heterogeneous scintillation counters as were described in Section II.F for beta radiation [48,50]. While this may be convenient in laboratories set up for beta detection, it is subject to the back pressure and memory effect problems common to heterogeneous detection.

The principle of Cerenkov radiation is applied using a Teflon-tubing flow cell surrounded by a solid scintillator [28,29]. This eliminates memory effects and makes back pressure negligibly low. This, like the method above, can also be used for high-energy beta radiation.

On-line gamma radiation may also be detected by passing a Teflon-tubing flow cell through a drilled sodium-iodide [51-53] or geranium crystal [51].

III. INFRARED DETECTION

A. Overview

1. IR Spectroscopy Infrared spectrometry is a detection method of much interest to chromatographers because it is information rich and nearly universal. The topic has been reviewed [54-59,120]. Infrared spectra give information on functional groups


such as carbonyls, amides, and hydrocarbon skeletons for all organic molecules and many inorganic ones. Molecules that cannot be detected by UV or refractive index are often easily detected by IR. The universality of this detection technique, while attractive for solute identification, causes problems in that all solvents used as HPLC mobile phases absorb in this region, which causes some wavelengths to be undetectable.

Infrared radiation is electromagnetic radiation with wavelengths between 0.7 and 500 μπι (wavenumbers 14,000 to 20 cm"1). It is normally broken down into three regions: near IR (12,000 to 4000 cm"1), mid IR (4000 to 650 cm"1) and far IR (667 to 10 cm"1) [60]. The mid IR region is further broken down into the group frequency region (4000 to 1300 cm"1) and the fingerprint region (1300 to 650 cm"1).

Although the near IR region has been utilized for HPLC detection [61-66], the mid IR region is most often of interest and is the only region covered here. Likewise, IR instrumentation will not be covered here; interested readers should consult an instrumental chemistry textbook such as Willard et al. [60] or the review article by McDonald [67]. For more detail of Fourier transform instruments consult Roush and McGrattan [68], Combellas et al. [69], Pattacini et al. [70], or Griffiths and de Haseth [71]. Interfaces and general instrumental considerations only will be discussed here.

The first general instrumental consideration is the choice between a Fourier transform infrared spectrometer (FTIR) and a conventional IR spectrometer. FTIRs have a higher energy throughput, a significantly better signal-to-noise ratio, and better sensitivity than conventional instruments. In addition, FTIRs can scan and store entire spectra rather than only one or two frequencies. While work has been done on conventional IRs, interest in the early 1980s turned to FTIR instruments and, because of the wealth of information they provide, they are for the most part the only instruments used for HPLC-IR today.

When IR detection is done on an HPLC analyte, interferograms are normally recorded and saved. This allows one to later see the analytes' spectra to aid in their identification. It also allows a Gram-Schmidt reconstruction to be done on the data to obtain a chromatogram where all components are not necessarily monitored at the same wavelength.

A Gram-Schmidt reconstruction uses the Gram-Schmidt orthogonalization process to establish a basis set that represents background signal. This is removed from all subsequent interferograms and the total infrared absorbance over the IR spectral range is determined and transformed into a chromatogram [72]. Other methods of chromatogram reconstructions have been discussed by Wang et al. [73]. More recently, Redmond et al. [122] have discussed the application of Kaiman filtering after Gram-Schmidt orthogonalization to enhance signal-to-noise ratios.

2. Detection Limits and Resolution The detection limit for a given compound depends on its molar absorption coefficient (Section III.A.3), the cell thickness, the Chromatographie peak volume, and the amount of spectral information necessary.

More absorptive peaks will be available at lower concentrations than less


absorptive ones. This was demonstrated by Gagel et al. [74] who report that at wavenumber 1678 the C = 0 stretching frequency of pheneanthrenequinone is detectable down to a 16-ng injection. To obtain a spectrum showing all useful characteristic absorbances, however, a 31-ng injection is needed.

Detection limits of HPLC/IR tend to be somewhat higher than that reported by Gagel, typically in the 100 ng to 1 ^g region.

Resolution in HPLC-IR is maximized by using systems with little or no dead volume, to prevent mixing outside the column. Another potential source of extra-column mixing is on the deposition surface of solvent elimination systems (see Section III.C.l).

One benefit of FTIR is that components not totally resolved on the column can be subtracted from each other to mathematically obtain more reliable information. Methods for this are discussed by Combellas et al. [69], Vidrine [75], and Mulcahey and Taylor [119].

3. Quantitation Infrared quantitation is done using Beer's law (absorbance equals molar absorption coefficient times concentration times path length), where the molar absorption coefficient is determined empirically. A series of known concentration solutions are injected and the peak heights are recorded. A plot of peak height versus concentration yields a slope equal to the path length times the molar absorption coefficient. Quantitation is accurate only for the concentrations found in the linear portion of the graph.

The peak chosen should be reasonably isolated so that the baseline can be clearly defined and be intense so that peak heights can be easily and accurately measured. It is also important to clearly establish that the chosen peak fits Beer's law. This may not be the case for a variety of reasons including hydrogen bonding and the presence of highly absorbing impurities.

The instrument used for quantitation must display a high signal-to-noise ratio, low drift, and a steady baseline.

4. Flow-Through versus Solvent-Elimination Detection It was stated above that the fact that an IR is a nearly universal detector causes problems. Standard IR solvents such as tribromomethane, triiodomethane, carbon disulfide, chloroform, and carbon tetrachloride are seldom used as HPLC solvents. Common HPLC solvents, particularly those used for reverse phase such as water, methanol, and acetonitrile absorb strongly in large sections of the mid-IR region, making them poor for use in IR detection. Normal-phase solvents may also absorb strongly, but the regions are smaller and can often be worked around.

Two types of detection are therefore used: flow-through and solvent elimination. Flow-through detection, used mainly for normal-phase chromatography, runs the effluent through a flow cell column without removing any mobile phase. Solvent elimination may be used with normal or reverse-phase chromatography. It involves the removal of solvent and the deposition of solute onto an appropriate substrate. Flow-through detection gives real-time analysis, solvent


elimination usually does not. These detection types will be discussed in Sections B and C.

5. Post-Column Preparations Normally when effluent leaves a column it is sent directly to a flow cell or to a solvent removal system, but this is not always the case. Some novel approaches have been used to remove water in reverse-phase systems to either facilitate evaporation or to allow the use of a flow cell.

One approach has been to extract the solutes out of the water with a solvent such as dichloromethane [76], deuterochloroform [77], carbon tetrachloride [78,79], or chloroform [79]. In the first case extraction was followed by solvent evaporation; in the other cases a flow cell was used.

The extractions were done by adding solvent to effluent and separating the phases using differential pressure across a membrane separator. The membranes were constructed initially with a single layer of polytetrafluoroethylene [79] and later expanded to three layers with different pore sizes [77,78].

The extraction solvents should be carefully chosen to extract all analytes. Shah and Taylor [77] compared carbon tetrachloride, chloroform, and deuterochloroform as extraction solvents for analgesics (acetaminophen, caffeine, sali-cylamide, aspirin, and phenacetin) and found a great variance in the percentage of analyte extracted. This variance was seen both between solvents and between analytes in the same solvent. Carbon tetrachloride proved to be the poorest extraction solvent, extracting no more than 18% of any analyte. Deuterochloroform and chloroform extracted 85-90% of caffeine, aspirin, and phenacetin; 50-55% of salicylamide; and 20-35% of acetaminophen. The main problem with the low percentages of acetaminophen and salicylamide extracted is the corresponding increase in the detection limit.

A second approach to water removal is that used by Kalasinsky et al. [80,81] prior to solvent evaporation. They reacted the eluate with 2,2-dimethoxyacetone in the presence of an acid catalyst to produce essentially quantitative amounts of methanol and acetone. These products are more volatile and hence more easily removed than water.

B. Flow-Through Detection

1. Solvents and Path Lengths Flow-through IR detection refers to cases where the mobile phase is not removed, but rather, the eluate is sent directly through the detector. It is therefore necessary to match the solute and solvent so that the important solute peaks fall in solvent windows.

Solvent windows are wavelength regions where the solvent transmits significant amounts of IR radiation. Transmittance of 30% is usually considered "significant," but amounts as low as 5% [82,83] can sometimes be used. Areas falling below this are considered opaque and, even with spectral subtraction, may not be used to gain information about the solute. For a simple mathematical


treatment of this see Johnson and Taylor [84]. Figure 9-5 shows 75% transmission windows for several common solvents.

The size of solvent windows depends on the solvent and the detector path length. Using the same cell thickness, reverse-phase solvents such as water, acetonitrile, and methanol have much larger opaque areas than normal-phase solvents such as hexane. Deuterated solvents, although expensive, are sometimes used, particularly in micro-HPLC, because they give larger solvent windows and may give better separations. This has been reviewed [85]. Deuterated solvents studied include heavy water [86-89], deuteriobenzene [87], deutero-

Acetone

Acetonitrile

Benzene

Carbon disulfide

Carbon tetrachloride

Chloroform

Cyclohexane

Deuterium oxide

Isopropyl alcohol

Methyl cyclopcntanc

Tetrachloroethylene

Wave number, cm 3600 3000 2000 1800 1600 1400 1200 1100 1000 900 800 700

I . , ■ 1 . . ■ . I . I ■ 1 ■ I . 1 . 1 ■ 1 ■ 1 ■ I ■ 1 ■ mm

0.1 ZML vmw/MWAWM/m w 3100 2900 1800

im: IH: -Ε35Γ 117011001080 910 830

JEL 3700 3500 2350 2250 1500 1350 1060 1030 930910

0.1 L IE! "JL 3100 3000

1.0 L ~vm 1820 1800 1490 1450

WMfa 1050 1020 680

ZSZL 2340 2100

0.1 C

Ί.0 C ZSL·

1640 1385 Β0Μ

875 845

2200 2140 1595 1460

1610 1500 12701200 1020 960 860 0.1 C W/M

1.0 C

0.1 C

o.i C o.i C 0.1

0.1

ZWl _0L W/A ΨΜΗΓ 3090 2980 2440 2380 1555 1410 12901155 940 910 860

820 720

3020 3000 1240 1200 805

3000 2850 1480 1430 910 850

2780 2200 Υ//////Λ

3600 3200

1280 1160 ΊΖΓ

3000 2800

1540 1090 990 960 830 VIWMWA

Z52L

i.o C 1480 1440 1390 1350

El 980 960

1370 1340 118010901015 0.1 C

935 875 820 745 2, 2, 4-Trimethylpentane 0.1 I W/////A

3150 2700 0.01 1 V/y/MWA

V/MWM ΊΜ~ 1430 1130 995 925

Water Y////A WMW/M/M 3650 2930 1750 1580 930

J_ JL J_ _L _L 5 6 7 8

Wavelength, μπι

10 11 12 13

FIG. 9-5 Transmission characteristics of selected solvents. The material is considered transparent if the transmittance is 75% or greater. Solvent thickness is given in millimeters. (Willard, H., Merritt, L., Dean, J., Settle, F. "Instrumental Methods of Analysis," 6th ed. Van Nostrand Co. Reprinted by permission of Wadsworth, Inc. Copyright 1981 by Litton Educational Publishing Inc.)


methanol [86,89], 90% deuteroacetonitrile with 10% heavy water [88], and deuterochloroform with a deuteromethanol modifier [90]. In addition, deutero-chloroform has been used as a post-column extraction solvent [77].

Freon 113 (l,l,2-trichloro-l,2,2-trifluoroethane) has also been used because of its IR spectral windows [91].

It was stated above that solvent windows are a factor of detector path length. This is because solvent windows are defined by percent transmission, and percent transmission is a function of path length, as described by Beer's law (Section III.A.3). A decrease in path length causes a decrease in the number of solvent molecules in the light path, which in turn causes a decrease in light absorption and an increase in light transmission. Analyte molecules dissolved in the solvent also experience a decrease in path length and therefore a decrease in absorption, which causes poorer detection limits.

The actual path length selected must be a compromise. A thick path length is desired for maximum sensitivity and a thin path length is desired for the largest solvent windows. A path length is therefore selected that is as wide as possible without obstructing the regions of interest. See Conroy et al. [76] and Griffiths et al. [57] for a further discussion on this.

Flow cells as thick as one millimeter can be used for mobile phases with large transparent regions such as chloroform, but a path length of 25 micrometers or less is necessary for solvents containing high percentages of water [92].

2. Advantages The advantages of flow-through detection over solvent elimination techniques include

Real-time analysis Simpler interfacing No loss of volatile components No thermal degradation of solutes

Interfacing usually consists of a piece of tubing connecting the HPLC column to the flow cell. The column may be physically located in the optical bench to minimize the length of tubing. A direct connection between a flow cell and a microbore column has been described [113].

3. Disadvantages Disadvantages of flow-through detection include

Opaque solvent regions Preferably a mobile phase with large solvent windows will be used, but opaque areas occur for all solvents, so some information is lost.

EJutions Spectral subtraction becomes either very mathematically complicated or impossible if an elution is used.

Reverse phase solvents These cannot easily be used due to large opaque regions.


4. Flow Cells Choosing the path length for a flow cell was discussed above. The other flow cell characteristics to be considered are construction materials and construction geometry.

The flow cell should be made of a material that is compatible with all solvents it may come in contact with; it should have a low refractive index and a low cutoff wave number. Table 9-3 below lists properties of various compounds used in IR flow cells.

Parallel plates are normally used. Johnson and Taylor [84] introduced a cylindrical flow cell with zero dead volume and reported improved detection limits over parallel plate cells. Their test compounds were tert-butyl phenols and cyclohexyl acetate.

Sabo et al. [93] used a cylindrical internal reflectance cell for attenuated total reflectance analysis of aqueous eluates. This provides a short path length for acceptable solvent windows, but the sensitivity is not sufficient for many applications.

C. Solvent Elimination

1. General Description and Interfaces Solvent elimination may be used for reverse-phase or for normal-phase systems. This topic has been reviewed by Griffiths and Conroy [94]. The goals of solvent elimination techniques include

Total solvent removal The only peaks present should be those of the analyte.

Quantitative deposition All analyte should be deposited on the designated surface. Solute lost during nebulization increases the detection limit.

TABLE 9-3

Compound

NaCl KBr CsBr Csl ZnSe AgCl BaF2

Properties of Compound:

Solubility in water

Soluble

Insoluble

Barely soluble

s Used in IR Cells

Refractive index

(at 2000 cm- 1)

1.52 1.53 1.67 1.74 2.5 2.0 1.45

Cutoff wavenumber

(cm"1)

650 400 250 200 500 450 850

Reprinted from Combellas, C , Bayart, H., Jasse, B., Caude, M., Rosset, R. (1983). /. Chromatogr. 259, 211-225. Courtesy of Elsevier Science Publishers.


These can be lost during mobile-phase removal, particularly when heat is used. This is to obtain maximum sensitivity. Decreasing spot size or bandwidth allows the IR beam to be more concentrated, which effectively increases path length. Solvent removal should occur at the same rate as it leaves the column. If removal is too slow, spot spreading takes place which reduces sensitivity and, if spots begin to mix, reduces resolution. If solvent removal is too rapid, deposition will occur too soon, potentially clogging the deposition system.

Solvent elimination techniques use a solvent removal system and a deposition substrate. Solvent removal and solute deposition are performed either together or in rapid succession. The deposition substrate (see Section II.C.4) is continuously moved in a circular or linear manner depending on the substrate employed. Solute may be deposited in spots or in a narrow linear band. After the HPLC run, the substrate is moved to an IR and again moved in a circular or linear manner to obtain a chromatogram.

The most common solvent removal systems are nebulizers. Several types have been used including ultrasonics [95,121]; monodisperse aerosol generators (MAGIC) [92,96-101]; and ones for gradients, which can be programmed to change temperature as the mobile phase changes [74,102].

MAGIC-HPLC/FTIR interfaces were first developed by Willoughby and Browner [103]. The interface consists of three chambers: a desolvation chamber, a first momentum separator chamber, and a second momentum separator chamber [99,103]. The desolvation chamber uses helium to disperse the eluate. The momentum chambers remove mobile phase and helium by vacuum pumps. The interface has been shown to remove solvents of up to 100% water at room temperature with the exception of a residual trace, probably trapped in the matrix. Spot sizes as small as 0.44 mm can be obtained.

Gagel and Biemann [74] built a nebulizer that uses a mixing tee to combine nitrogen and the eluate. This is passed through an envelope of heated nitrogen and sprayed onto the desired surface, here a reflective mirror. The nitrogen was heated with a variable transformer that could be changed throughout a run to handle gradients. The method had not been perfected because cooling was by convection only.

Mobile phase can also be removed by gentle heating rather than nebuliza-tion. This was done by Conroy et al. [104] using preheated cups of KBr. Each 2-mm diameter cup received one drop of 8 to 10 μΐ. as it came through a capillary tube after exiting a microbore column. The mobile phase, 2% methanol in

Retention of volatile and thermally labile molecules

Deposition in small spots or narrow bands

Proper rate of solvent removal


hexane, was found to evaporate immediately so no additional heating was necessary.

Gentle heating alone is usually used only for normal-phase systems. A few additional comments on spot size and bandwidth are now included.

Deposition spot size or bandwidth is determined by the solvent removal system and the deposition surface. Smoother surfaces tend to see more spreading than coarser ones. It is desirable to have spots less than 0.02 mm2 [105], but this is usually not attainable. 100 and 250 μπι spots on KCl have been reported by Fräser et al. [106,107]. Spot size may be limited by depositing the eluate into cups [80,104,108-111], but this is time consuming because of the necessary setup and clean-up involved.

2. Advantages The advantages of solvent elimination include

No information is lost due to solvent opacity. Volatile buffers are removed [92]. Normal or reverse-phase solvents may be used. Gradient elution can be used. This may require programmed heating and

cooling of the nebulizer, particularly for reverse phase. Sensitivity may be enhanced by changing the FTIR mirror speed in crucial

areas. This is possible since real-time analysis is not used.

3. Disadvantages The main problems and considerations of solvent elimination include

System complexity The systems require more complex interfaces than flow cell systems.

Loss of volatile components Nebulizers and heating systems may remove volatile analyte molecules. In addition, if heat is employed, thermally labile molecules may be degraded.

Residual solvents Mobile phase may be trapped in the matrix, so some solvent bands may appear in the spectra.

Atmospheric gases Water vapor and other atmospheric gases may show up if the system is not designed to avoid it [108].

Fumes Care must be given to remove toxic mobile-phase fumes in an acceptable manner.

Deposition surfaces Unevenness of deposition surfaces can cause interferogram intensities to vary. This causes reconstruction to give erroneous results [80,104] and spectral distortions [75].

Analysis usually not real time Frequently the deposition surface must be moved from the HPLC to the IR.


Solvent removal rate Solvent removal must be rapid enough to avoid resolution or sensitivity loss, but not so rapid that solute accumulates in the nebulizer.

4. Deposition Surfaces Surfaces used to deposit mobile phase-eliminated analytes vary according to the mode of IR. In general the surfaces should be impervious to any remaining mobile phase, should not absorb any IR radiation, and should satisfy the requirements for the detection mode used.

KC1 is a frequent choice for systems employing diffuse reflectance. Kalasin-sky et al. [80] formed "trains" or "troughs" with KC1 such that positioning in a diffuse reflectance cell is repeatable. Conroy et al. [104] used it in 2-mm cups. In both these cases better results were obtained if the KC1 was flattened by applying pressure rather than used as a loose powder. It should be kept in mind that KC1 and KBr are water soluble and should be avoided if residual water may be present.

KC1 has also been used for diffuse transmittance. Fräser et al. [106] have studied the effect of diffuse reflectance versus diffuse transmittance of phenan-threnequinone in KC1. They found that when dealing with volatile mobile phases diffuse reflectance is most successful, and when dealing with less volatile mobile phases diffuse transmittance is preferred.

Diamond powder may also be used for diffuse reflectance [95,112], which is attractive for reverse phase because it is insoluble in water.

Fujimoto et al. [114] developed a stainless steel wire net, which they used as a substrate for transmission detection. This was attractive because it allowed good air flow for efficient solvent removal.

KBr windows (MAGIC) [92,99] or plates (buffer memory) [115,116] can be used for transmittance or absorbance.

The reflectance-absorbance mode of detection requires a reflective surface. Germanium or zinc selenide coated on aluminum, copper, or gold is one reflective surface [102,117] that may be used. An aluminum mirror is another [74]. The aluminum mirror alone, however, when used to collect thin deposits, may cause an artifact in the spectral distribution skewing the chromatogram by favoring high-frequency absorptions [102]. Coated surfaces are therefore often preferred.

IV. LIGHT-SCATTERING DETECTION

Light-scattering detection utilizes a wavelength of light that is not absorbed by the eluate. The light comes in contact with the sample and is scattered. This scattering is detected at a given angle or angles from the incident light to obtain information about the solute.

This section is broken down into two types of light scattering. The first, evaporative light scattering, volatilizes the solvent and detects the particles remaining as solids or oils. The second, solution light scattering, utilizes differ-


ences in Brownian motion or dielectric constant between solute and solvent. An additional method of on-line HPLC light-scattering detection has been reported by Jorgenson et al. [123] for the study of lipids. They used ammonium sulfate for post-column solute precipitation, and then measured the light scattered by the precipitate. This will not be discussed.

A. Evaporative Light-Scattering Detectors

1. Overview Evaporative light-scattering detectors are also called aerosol light-scattering detectors or mass detectors (although they are not true mass detectors). They are based on the principle that scattering of light depends on particle size. They are mainly used for biological molecules such as triglycerides, fatty acid esters, and steroids. These detectors have recently started to look promising for detecting adulteration of expensive edible oils and fats by less expensive oils [124,125].

Before continuing, it may be useful to describe what is meant by a "mass detector." A mass detector is a universal detector, that is, one that detects all compounds, regardless of physical properties and chemical composition. Beer's law and extinction coefficients do not apply. Peak area is determined only by the mass of material in the peak. Evaporative light-scattering detectors may show some tendencies toward this ideal, particularly for similar types of compounds, but they cannot be considered true mass detectors.

Evaporative light-scattering detectors consist of a nebulizer that aerosalizes the eluate, a drift tube to vaporize the solvent, and a light-scattering cell where the scattering takes place and is detected (Fig. 9-6). Detection limits are about

LC Eluate Nebulizer Gas

Light Source

FIG. 9-6 Block diagram for an HPLC light-scattering detector.


100 ng for glucose [126], 30-100 ng for triglycerides [127] and 1.5 μ% for prednisone [128].

2. Theory

a. Light interactions When particles, used here to mean solid particles or nonvolatile oils, are hit with a beam of light, several things may happen. The light may be absorbed, refracted, reflected, Rayleigh scattered, or Mie scattered.

If absorption takes place, a detector other than light scattering should be selected, or a different wavelength should be used. Light-scattering detection cannot be used if the analyte particles or solvent vapors absorb the light.

Reflection and refraction always occur together, and the sum of their intensities equals the intensity of the incident light. These prevail when the wavelength of light approaches the particle size [129,130].

Mie scattering occurs when the ratio of particle diameter to the wavelength of light is greater than 0.1. Rayleigh scattering occurs when the ratio is less than 0.1 [131]. These numbers are approximate and a transition region does exist. Scattering theory will not be discussed here; it will suffice to say that Mie scattering is more complex than Rayleigh scattering. For more on Mie theory see references [132,133].

Detector response (peak area) obtained from a given sample has been described as linear [134], sigmoidal [130,132], and exponential [127,131]. The last case may be expressed as

D = amb

where D is peak area, a and b are numerical coefficients, and m is sample mass. The data are plotted as log D versus log m to obtain a graph that has a large

linear region with a slope b and an ordinate a. This region can be used for quantitation. The slope b, which tends to be similar for similar compounds, falls between 1 and 2; 2 is the limiting value for Rayleigh scattering [135]. Mourey and Oppenheimer [132] predicted a response curve for the log-log graph that contained both linear and nonlinear regions, and which would give a sigmoidal response when plotted on linear axes.

b. Droplet size One property that is very important in determining the scattering that takes place is particle size. As was shown above, the relationship between this and light wavelength determines the type of scattering observed. "Droplets" become "particles" in the drift tube by solvent vaporization. Particles are the nonvolatile portion of the eluate. They may be oils or solids.

Particle size is determined by the droplet size that leaves the nebulizer as described by the equation [132]

XP = X(c/d)1/3

where XP is particle diameter leaving the drift tube, X is droplet size entering the drift tube, c is solute concentration, and d is solute density.

It is evident here that droplet size and solute properties determine the size of the particles seen by the light-scattering cell.


The droplet size is determined by the nebulizer and the eluate's properties, in particular surface tension, viscosity, and density [136]. An equation has been formulated to describe the relationship between droplet size and solvent parameters called the Nukiyama and Tanasawa equation [137] (or see [132] and references therein). This equation is complex, and while it does not always hold [131], it can often be quite useful.

Equations have also been formulated to determine droplet size distribution [132,138,139] and solvent vaporization time [130], but they will not be discussed here.

3. Instrument Design As was described above, evaporative light scattering involves nebulization of the column eluate to form an aerosol, followed by solvent vaporization in a drift tube to produce an analyte cloud and then detection in a light-scattering cell (Fig. 9-6). Solvent is not removed, it simply does not scatter light and therefore does not register in the light-scattering cell. The three parts of the system to be discussed are the nebulizer, the drift tube, and the light-scattering cell.

a. The nebulizer The nebulizer is normally interfaced directly to the LC column. It combines the eluate with a stream of gas to produce an aerosol. For the best sensitivity, the aerosol produced will have large droplets and be of low polydispersity. The nebulizer properties that are adjusted to obtain these are the gas flow rate and the eluate flow rate [140].

As the gas flow rate is increased, both the signal and the noise levels decrease markedly [141]. At very low gas flow rates, nebulization is improper and droplets are produced that are too large for the drift tube to vaporize. The result is a large noise level. In general, a low gas flow rate is desired, just high enough for proper nebulizer operation. This will produce the desired large droplets without making ones too large for the drift tube to handle. It is important that the gas flow rate be stable for accurate quantitation.

As the eluate flow rate increases from 0.5 to 3 mL/min, Robinson et al. [142] found that detector response decreases. The curve produced was sigmoidal with the steepest slope between 1 and 2 mL/min. This clearly demonstrates the need for flow rates to be consistent between runs.

b. Drift tube Volatile components of the aerosol produced by the nebulizer are evaporated in the drift tube to produce nonvolatile particles in solvent vapors. Ideally, the heated drift tube will rapidly evaporate all solvent without any solute vaporization, droplet coagulation, or particle precipitation.

If solvent removal is incomplete, detector noise will increase. If very large droplets reach the light-scattering cell, they will be seen as spikes, which can be smoothed by software but at the expense of sensitivity.

Solute vaporization may occur if the drift tube temperature is too high or the solutes are too volatile. This will decrease sensitivity. For best results, solvents should be much more volatile than solutes.

Droplet coagulation is the joining of aerosol droplets in the drift tube. It can cause incomplete solvent removal and detector signal spiking. Precipitation


causes a decrease in sensitivity. It, like coagulation, increases if the nebulizer gas pressure is too low.

The drift tube should be wide enough, long enough, and hot enough to ensure complete and rapid solvent removal. Its outlet into the light-scattering cell should be shaped to send all the particles past the detector window.

c. Light-scattering cell The particle cloud leaves the drift tube and enters into a light-scattering cell. Laser light normally around 632 nm is shined into the cell through a window, scattered by the analyte, and detected at an angle to the incident light. Polarized or nonpolarized light may be used [135].

The detector should be constructed so that material in the particle cloud will not stick to the window and so that fumes are properly vented. In addition, a light horn to trap and dissipated nonscattered light should be placed opposite the light source.

4. Considerations

a. Noise Noise sources in evaporative light-scattering detection include

Residual solids in the solvents High-quality HPLC solvents should be used, but even these typically contain 5 to 10 ppm dry residue [143], which should be compensated for electronically.

Dust from column packing material This can be removed by using a tight frit at the end of the column.

Particles adhering to the optical This can be lessened by the detector window design [140].

Oil from the gas compressor Incomplete solvent vaporization This can originate from a gas flow

rate that is too slow or a drift tube temperature that is too low.

Diffraction on light-scattering window

b. Solvents One nice feature about evaporative light-scattering detection is that a wide range of solvents can be used, including ones such as acetone and chloroform that are not good for UV detection. Solvent requirements include that they be

Significantly more volatile than the solute. Clean, that is, with only a very small amount of dry residue. This require

ment also limits the use of nonvolatile buffers. Nonabsorbing at the wavelength used. Nonexplosive [144].

c. Gradient elution When gradient elution is used, baseline drift is not seen, providing the solvents remain clean and totally volatilized. The absence of baseline drift in itself does not mean it can be used. Sensitivities change in


solvent gradients, due mainly to changes in droplet size. These changes are caused by changes in eluate properties such as surface tension, viscosity, and density. If droplet size remained the same, gradient sensitivity would show little change [128].

Solvent gradients can be used, but it is important to quantitate the method for the particular gradient and compounds intended for use. In general, linear gradients work the best [145].

d. Quantitation These detectors are not true mass detectors. They must be calibrated for each compound. Calibration should be done using the gas flow rate, solvent flow rate, solvent, temperature, and physical system set-up that will be used for quantitative runs.

B. Solution Light-Scattering Detectors

Light-scattering detectors for solutions are normally used in sequence with UV and/or refractive index detectors. They are mainly used for the characterization of synthetic, inorganic, and biopolymers.

Two types of light-scattering detectors are used for solutions. One is called photon correlation spectroscopy and the other laser light-scattering photometry.

1. Photon Correlation Spectroscopy Photon correlation spectroscopy, also called dynamic light scattering or quasi-elastic light scattering, uses the fact that the speed at which molecules move in solution is dependent on their hydrodynamic radius. Larger molecules move slower than smaller ones. Coherent light is scattered by these molecules as they move relative to each other, causing a pattern of constructive and destructive interference. The interference is measured and used to calculate the hydrodynamic radius, from which uniformity and molecular weight are calculated. An excellent review article on the theory of this technique (for static systems) is given by Phillies [146]. Shorter summaries of the theory [147], including online applications have been presented [148], as have details on the use of this detector type for HPLC [149-152].

2. Laser Light-Scattering Photometry Laser light-scattering photometry is used to determine weight-average molecular weight and molecular size. It uses principles of Rayleigh scattering and the intensity of scattered light as a function of scattering angle. An excellent review of the theory and its applications to biomolecules has been given by Stuting et al. [153]. Takagi has reviewed its use for proteins [154]. Nicolai et al. [155] have combined LALLS (low-angle laser light-scattering) with refractive index to characterize DNA proteins eluting in gel permeation chromatography, and Maezawa and Takagi [156] give a method of using LALLS in conjunction with refractive index and UV to determine molecular weights of glycoproteins. Krull et al. combined detection techniques to detect dimers and aggregates of bovine alkaline phosphatase [157,158]. They then extended the technique to other


enzymes and proteins in a gradient elution system [159,160]. Flapper et al. [161] compared the molecular weight determinations from HPLC-LALLS and gel permeation chromatography to determinations from ultracentrifugation for serum proteins.

V. OPTICAL ACTIVITY

A. Introduction

Optical activity (OA) is of interest because it is often an indication of biological activity. OA detectors are selective and therefore a good choice for complex mixtures where some components possess optical activity. OA detectors for HPLC have found applications in the analysis of drugs, carbohydrates, steroids, nucleosides, and enzymes [162].

Two excellent review articles have been written. The first, by Purdie and Swallows [163], discusses both conventional and on line applications of OA detection. The second, by Lloyd and Goodall [164] discusses HPLC OA detectors in detail. It gives excellent discussions on laser noise and quantitation schemes for incompletely resolved chiral mixtures.

Three methods are used to measure optical activity: polarimetry, circular dichorism, and optical rotary dispersion (ORD). Polarimetry and ORD measure the rotation of a plane polarized when it passes through a sample. Polarimetry measures one or more wavelengths, while ORD scans a spectrum. ORD presents problems in baseline definition and specificity in differentiation [163]. It is not discussed here because it is seldom if ever used in HPLC. Circular dichroism is the difference in absorption between left and right circularly polarized light. It and polarimetry are discussed below.

B. Polarimetry

Polarimetry is the measurement of the rotation of plane polarized light as it passes through a medium. The amount that a given compound rotates light is dependent on the concentration, path length, and rotary strength of the compound, the observation wavelength, the solvent, the temperature, and the pH. Because of the number of factors involved, literature values are often not available for the conditions used in HPLC.

Typically polarimetric data is reported as a specific rotation (a) defined as the number of degrees a compound rotates light in a 1 dm length of tube at a sample concentration of 1 g/cc in water. Specific rotations are reported with the wavelength (usually the sodium D line for conventional polarimeters) and temperature used for the measurements. Polarimeters respond equally well to both absorbing and nonabsorbing analytes, which is contrary to circular dichroism where only absorbing analytes are detected.

On-line polarimeters are less sensitive than UV detectors and more sensitive than refractive index ones. Limits of detection are typically in the nanogram range: 250 ng for limonene [165], 50 ng for proline, 120 ng for threonine, and 2 μg for tyrosine and phenylalanine [166]. Detection limits may be improved


by using lower wavelengths and through derivatization. In the latter case, it must be verified that no racimization has taken place.

1. Instrumentation The first polarimetry detector for HPLC was described by Yeung et al. in 1980 [167]. Earlier on-line polarimetry detection had been used and an extension to HPLC had been suggested [168]. Commercial detectors have recently been introduced [169,170].

Polarimetric HPLC detection requires small flow cell volumes for optimal resolution, and microdegree sensitivity for good detection limits. Conventional polarimeters are not sensitive enough, so adaptations had to be made.

The basic design of an HPLC polarimeter includes a light source, a lens to focus the light, a polarimeter, a modulator, a flow cell, an analyzer, and a photodetector. The polarizer and the analyzer are normally Glan-Taylor prisms [167] or sheet polarizers [171]. The photodetector may be a silicon photodiode [172] or a photomultiplier [167]. The modulator is usually a Faraday cell used to provide a standard optical rotation. The light source and the flow cell are discussed below.

a. Light source Laser light is normally used in on-line polarimetry because it provides better power throughput, focusing superiority, and increased spectral purity over conventional light sources. A laser can provide microdegree measurements, while conventional sources provide millidegree measurements at best.

Two types of lasers are used, gas and diode. Gas lasers include argon ion lasers used at 458 nm [173], 488 nm [173] and 514 nm [174]; and HeNe lasers operated at 633 nm [166]. Diode lasers, which are in commercial instruments, are used at 780 nm [171] and 820 nm [169].

While lasers offer the advantages listed above, they present problems. The first is that they tend to be noisy and the second is that available wavelengths are limited.

Lasers exhibit both flicker noise and shot noise. Attempts to limit flicker noise or intensity instability include using very low depolarization, high-frequency modulation, balanced photodetectors, multiple polarizers, a combination of these, or a semiconductor diode laser [see reference 172 and references therein]. The most commonly used approaches are high-frequency modulation and diode lasers. One major source of shot noise was found to be an incompletely extinguished laser beam [167]. This problem was handled by Kuo and Yeung [175] by passing the laser light through a Pockels cell and then a slightly off axis Glan prism.

Wavelength limitations are a problem due to optical rotary dispersion effects. Short wavelengths tend to have higher rotary powers and therefore better mass sensitivities than longer ones; 488 nm offers approximately one-half and 820 nm offers approximately one-sixth the mass sensitivity of 365 nm [164]. No inexpensive stable UV lasers presently exist that are suitable for commercial polarimeter use [164]. Despite this limitation, lasers still offer more sensitivity than conventional light sources.


b. Flow cell Flow cells ideally are of long path length for maximum sensitivity and of small volume for maximum resolution. These are difficult to accomplish together. Cells 1 to 2.5 cm long with a volume of 8 to 20 μΐ. have been used [172,176], as have flow cells 10 cm long with a volume of 200 μΐ, [174] and 5 cm long with a 100-μΧ. volume [177]. Microbore cells as small as 1 cm have been reported with a volume of 1 μΐ, [178]. One commercial detector has a 50-mm path length with a 40-/xL volume [170], another has a volume of 18.5 μΐ, [169].

Flow cells may be straight bore or tapered. Lloyd et αΐ. [172] found tapered cells to be desirable for use in gradient chromatography because they reduced refractive index effects.

Cells must be carefully aligned to reduce light scattering and depolarization. Flow cell windows may cause birefringence (double refraction) and addi

tional light scattering. These can be lessened by placing the windows slightly off normal [175].

c. Problems and considerations include

Pump pulsations [176]

Dust particles [179] Refractive index changes [177]

Mobile phase gas [176]

Dirty check valves and seals [177]

Flow-induced birefringence [172]

Potential problems and considerations

These can be lessened with a pulse suppressor.

These may be seen during gradient elu-tion in a manner similar to that seen in UV detection. They cause laser beam def ocusing and can be avoided by carefully choosing gradients. Refractive index changes may also be seen during the elution of nonoptically active compounds as peaks that should not be there. These peaks are usually quite small and integrate to near zero. This can be eliminated by degassing solvents and leaving them under a positive pressure of helium. These should be checked and cleaned regularly. Swadesh demonstrated this by showing the change in baseline noise seen in the detection of camphor with and without check-valve soni-cation. Lloyd et al. reported artifacts occurring with varying flow rates in the analysis of amino acids. They believed these were due to flow-induced birefringence. The problem was eliminated by aligning the beam of polarization with the direction of flow.


Residual depolarization [175] Thermal variations While these are not as serious as in

refractive index detection [175], they can cause changes in the optical rotation of analytes. In addition, laser heating causes birefringence changes in the polarizing crystals and cell windows. These can be lessened by using lower laser powers [179].

Laser noise [175] Misaligned flow cells

2. Detection Application Types Polarimetric HPLC detection is normally used for one of five types of appli

cations.

1. Detection of chiral molecules in mixtures where only one enantiomer of any given compound is present.

2. Detection in systems where both enantiomers of a compound are present and a partial or total chiral separation has taken place.

3. Detection in systems where both enantiomers of a given compound are present and no chiral separation has taken place.

4. Detection of optically inactive compounds through the use of an optically active mobile phase (indirect polarimetry).

5. As an absorption detector.

Each of these is discussed below. In the first application, the object is to separate the various optically active

molecules from each other. Typically the analyte mixture is from a biological source. Applications include the analysis of free and esterified cholesterol in human serum [179], carbohydrates in urine [175], menthyl acetate in peppermint oils [180], sugars in complex mixtures [169], steroid mixtures [162], nucle-oside mixtures [162], shale oil extracts [174], and L-amino acid mixtures [177,181].

Reitsma et al. [177,181] used a polarimetric HPLC detector to demonstrate the separation of L-amino acids (Fig. 9-7). To increase rotary power and thereby improve detection limits, the dansyl [l-(dimethylamino)naphthalene-5-sulfo-nyl] chloride derivatives were used. This was found to increase the specific rotation of many, but not all of the derivatized compounds. From Fig. 9-7 it can be seen that all the dansyl amino acids gave peaks, including glycine, which is not optically active. This peak, which is attributed to refractive index changes caused by glycine elution, gives a near zero integration and is therefore of no concern.

In this type of separation, the concentration of a given analyte in g/mL has been calculated using specific rotations of the sodium D line (589.3 nm). This was employed by Kuo and Yeung in the analysis of carbohydrates in urine [175]. Their optical activity detector used an argon laser at 488 nm and their eluent was water. Concentration was determined by the ratio al [a] where a is the


OA

Time (minutes)

FIG. 9-7 Optical activity chromatogram of a dansyl-L-amino mixture: 1, ASP; 2, GLU; 3, HYP; 4, ASN; 5, SER; 6, THR; 7, GLY; 8, ALA; 9, PRO; 10, VAL; 11, NVAL; 12, MET; 13, ILE; 14, LEU; 15, TRP; 16, PHE; 17, CYS. (Reprinted with permission from Reitsma, B., Yeung, E.; AnaJ. Chem. 59(7) 1059-1061. Copyright 1987 American Chemical Society.)

rotation determined on-line and [a] is the specific rotation of the Na D line. They found that the wavelengths were close enough so that the Na D line was a good estimation to use. In their later work with free and esterified cholesterol [179] using a laser at 514.5 nm and a tetrahydrofuran-water (76 : 24 v/v) mobile phase, they found an error of up to 10%.

Reitsma and Yeung [182] presented a method of determining peak purity using a UV detector in series with an OA detector. If the OA: UV ratio is constant throughout the elution of a given peak, peak purity is indicated.

The second type of separation in which OA detection is useful is one in which both enantiomers of a given compound are present and the object is to separate them. If a complete chiral separation has taken place, two peaks will show up on the OA Chromatogram. This was demonstrated by Shibukawa et al. [183] in the determination of free warfarin concentrations in serum albumin.

Often in mixtures containing both enantiomers, Chromatographie separation attempts provide only a partial chiral separation. In these cases a UV detector may show one peak while a polarimetric detector provides two fused peaks (a positive peak fused to a negative peak). This was clearly demonstrated for


a mixture of pseudoephedrine enantiomers [184] and a mixture of trans-1,2-diphenylcyclopropane enantiomers [185]. In cases involving partial chiral separations, a polarimetric detector is vital to determine if any separation has taken place, which in turn is vital in the development of chiral separation methods. Figure 9-8 illustrates the differences obtained using a UV/visible absorbance detector and an optical rotation detector.

Mannshreck et al. [186,187] designed a method for enantiomeric purity determinations in cases of incomplete chiral separations. They prepared a plot of optical rotation versus UV absorbance over the time a peak elutes. If some chiral separation has taken place, the initial portion of the graph will be linear indicating the elution of a single enantiomer. As Chromatographie conditions are changed, the linear portion of the graph can be used to determine if the separation was better or worse than under previous conditions. It also can be used to determine when a fraction should be collected to obtain a pure enantiomer.

The third polarimetric HPLC application is in the determination of enantiomeric ratios when no chiral separation has taken place. The HPLC serves to separate the analyte from interfering impurities. To determine enantiomeric ratios, a pure sample of each enantiomer is needed. A set of mixtures is prepared ranging from 0 to 100% of the L isomer (100 to 0% of the D isomer). Each mixture is run through the HPLC system and detected by two detectors. The first, usually a UV, responds to the total amount of analyte and the second is an OA detector. A standard graph is prepared using the ratio of OA to UV versus fraction of L (Fig. 9-9). This can then be compared with an unknown sample to determine enantiomeric purity [166,188]. If the compound of interest is not UV active, a refractive index detector can be used. The standard graph is then the ratio of OA to RI versus fraction L.

Reitsma and Yeung [166] demonstrated this technique for a series of amino acids. RI detection was used for proline and threonine and UV detection was used for phenylalanine (Fig. 9-9) and tyrosine. In all cases correlation coefficients were better than 0.99. Lloyd et al. [172] made similar determinations on tryptophan mixtures.

A variation of this technique was presented by Meinard et al. [189] in their work on the insecticide deltamethrin. Their main concern was the identification of diastereomers that form from photoisomerization. Polarimetric detection allowed diasteromer identification and chiral purity determinations of enantiomers.

The fourth polarimetric application is quite different from the first three. Here indirect polarimetry is used in a manner similar to that used by Mho and Yeung [190] for indirect fluorometry and Small and Miller for indirect UV detection [191].

The basis of these indirect methods is that the detector responds to a physical property of the eluent. A steady background signal is present when no analytes are present. When analytes are present, fewer eluent molecules are in the detector, hence the background signal is changed, usually decreased. This change is corrolated to a quantity.

In polarimetric detection, OA is the physical property of interest. A steady


Minutes

Minutes

FIG. 9-8 A simulated separation of enantiomers (the retention times differ by 0.10 minute). Thi upper panel illustrates the results obtained using a UV/visible absorbance detector, and the lowe panel is that of an optical rotation detector.


120H

ÜV 1

-120 - ^ . 1.0 0.5

Fraction L

FIG. 9-9 Enantiomeric ratio calibration curve for phenylalanine. The total amount injected is approximately 50 /xg. The exact amount is not needed for calculations, since the ratio of responses is plotted. (Reitsma, B., Yeung, E. (1986) /. Chromatogr. 362, 353-362. Courtesy of Elsevier Science Publishers.)

optical rotation background signal is produced so that when optically inactive analytes pass through the detector a decrease in the background signal is observed. If optically active analytes pass through the detector, a change in rotation will be seen providing that the specific rotation of the analyte is different from that of the mobile phase, which is almost always the case.

Indirect polarimetic detectors are nearly universal, as are refractive index ones. Limits of detection may be as low as 4 ng [192] injected material; below this, thermal noise becomes a problem. The nature of the analyte matters little since it is essentially the displacement of mobile-phase molecules being measured. Mobile phases with high specific rotations give lower detection limits than ones with smaller specific rotations. Solvent purity is not critical, impure solvents just cause some loss of sensitivity [193]. Due to the cost of chiral solvents, microbore chromatography is normally used.

Physically, indirect polarimetry is performed by manually rotating the analyzer to give a new zero. Electronic supression is unnecessary so there is no limit to the specific rotation of the mobile phase that can be used. This is contrary to indirect UV, where there is a limit to how highly absorbing an eluent can be used.

Indirect polarimetry has been demonstrated by Bobbitt and Yeung using ( - )2-methyl-l-butanol: acetonitrile 50 : 50 mixture [[a] = - 2.95°) as the eluent in the determination of dodecane [178]. They obtained a detection limit of 390 ng and predicted that if a mobile phase with [a] = 100° was used, a detection

0.0


limit of 12 ng could be achieved. This assumes that the baseline noise would not increase.

A method has been presented to obtain quantitative information about an analyte without identifying the analyte. This was described by Yeung for use with refractive index detection [194] and later extended to indirect polarimetric detection [178]. It will not be discussed here; interested readers should consult the references.

The fifth application of polarimetry in HPLC detection is absorption detection, which is an extension of indirect polarimetry. It is based on the fact that when an analyte absorbs radiation it heats up and expands. A chiral eluent is used, so this expansion causes a decrease in optical rotation. Absorption can be differentiated from simple indirect polarimetry by varying the laser power. A linear change in peak height with laser power will be observed for absorbing species, but not for nonabsorbing ones.

Absorption detection limits are 12 ng for dibutyl phthalate and 36 pg for N-methyl-o-nitroaniline [173]. Bobbitt and Yeung [173] in their work with N-methyl-o-nitroaniline showed that, as would be expected, the detection limit could be improved by choosing a wavelength that is as close as possible to the compound's absorption maximum. They obtained a detection limit of 7 ng at 488 nm and a limit of 36 pg at 458 nm.

C. Circular Dichroism

Circular dichroism (CD) measures the difference in absorption of left and right circularly polarized light. It requires an analyte with both asymmetry and an absorbing chromatophore, and is therefore more selective than polarimetry. Because there is no CD spectrum where there is no analyte absorption, a baseline is easily defined. A clear and concise discussion on HPLC-CD is given by Yeung [193].

No real-time, full-spectrum CD detectors are currently available for HPLC, but one would not be beyond development if the need arose [163]. Conventional CD, like conventional polarimetry, is not sensitive enough for HPLC use. Lasers are required for optimal sensitivity, which means that light source stability is important, as it is in polarimetry. Lasers present a problem in that they are not tunable to provide a scan of wavelengths as would be desirable for CD detection. The limit of detection for CD will always be a few orders of magnitude worse than that of UV absorption [195].

Applications have been presented that use conventional light sources. West-wood et al. [196] found a detection limit of 3 μg for L-tryptophan using a commercial CD instrument fitted with a flow cell. They monitored the eluate at 270 nm and stopped the flow to obtain a complete spectrum at points of interest. Drake et al. [197] adapted a UV instrument to obtain CD data for pavine enanti-omers. They selected the wavelength of the lowest frequency CD band maximum or the major CD band if more than one CD band is associated with the lowest energy absorption band. Salvadori et al. [198] presented an alternate method for choosing a CD band in their work with arylalkylcarbinols. They also discussed the determination of absolute configurations from HPLC-CD data.


As expected, much better detection limits have been obtained with the laser-based instruments. Synovec and Yeung [199] obtained detection limits of 38 ng and 5.6 ng respectively for conventional and microbore chromatography of ( + )-tris(ethylenediamine) cobalt(III). They used an argon laser at 488 nm.

Xu and Tran [200] found a detection limit of 7.2 ng for both enantiomers of the same cobalt complex. They developed an ultrasensitive thermal lens CD spectropolarimeter based on the difference in the amount of heat generated between left and right circularly polarized light. They used an argon ion laser for excitation and a He-Ne laser to produce a probe beam. Heat generated by sample absorption changed the probe beam intensity, which was detected by a photodiode.

An alternate CD method may be used for compounds that possess fluorscent properties. Synovec and Yeung [201] combined the selectivity of fluorescence and the selectivity of optical activity to produce a highly specific detector. Using a He-Cd laser at 325 nm they obtained a fluorescent-detected circular dichroism (FDCD) limit of detection of 168 pg for ( - )riboflavin. A problem arose in that an FDCD peak was also present for 4-methylumbelliferone, which is not optically active. This was attributed to its large molar absorptivity.

VI. OTHER DETECTORS

A. Element-Specific Detectors

Element-specific detectors quantify atoms of a given element without regard to their molecular association. These detectors can be divided into two broad classes, atomic absorption spectrometers (AAS) and atomic emission spectrometers (AES). They have received so much attention that it is not possible to do them justice here. A brief summary will therefore be given, followed by recent developments.

Both classes of detectors begin with the volatilization of the analyte solution received from the HPLC, followed by atomization to produce individual analyte atoms. These atoms are excited and either the absorption (AAS) or the emission (AES) of light is monitored. Within each class there is a variety of vaporization, atomization, and excitation techniques. For atomic emission, atomization techniques include inductively coupled plasma, atomic fluoresence [202], DC plasma, radio-frequency-discharge helium plasma [203], microwave induced plasma, and flame emission. For atomic absorption, flame and electrothermal (such as graphite furnace) atomizations are used.

These detectors have been reviewed Jewett and Brinckman [204]. More recently, Harrison and Rapsomanikis edited a book [205] dedicated to atomic spectroscopy interfaced with Chromatographie systems (GC, HPLC, SFC) for environmental analysis. Other reviews have been written [59,206-209] including ones on sample introduction techniques [210,211]. In addition, LaFreniere et al. [212] have described the ideal characteristics of on-line element specific detectors.


1. Atomic Emission Spectroscopy DC plasma-AES has found application in the analysis of many elements including Cr [213], As, Fe, Mn, Pt, and V [214]. One particularly interesting application is in the analysis of platinum antitumor agents in biological fluids [215] and bulk drug substances [216].

Inductively coupled plasma-AES has seen advances in interfacing via ther-mospray [217] and desolvation [218]. Thermal gradients have been used to avoid solvent gradients [219], and many elements have been detected simultaneously [220]. This technique has also been used in the determination of tetracycline binding in biological systems [221].

Galante et al. [222] studied the use of microwave-induced plasma-AES with replacement ion chromatography (RIC) and obtained detection limits of 30-300 ng for anions and 100-500 ng for cations. They also described the design of a flame emission spectrometer for use with RIC [223].

Flame photometry is an element-sensitive detection method used mainly for the analysis of phosphorus and sulfur. Kientz et al. [224] have reported success in analyzing nonvolatile organophosphorus acids. Detection limits of 0.5 to 2 ng were obtained with a linear range that spanned two orders of magnitude. They also described a preconcentration system that they used in the analysis of several monophosphate esters, including cyclic adenosine mono-phosphate [225]. In the analysis of sulfur, Okazaki et al. analyzed alkyl phenyl-thiocarbamates using an electrospray interface. They obtained a detection limit of 0.2 μg for hexanal thiosemicarbazone [226].

2. Atomic Absorption Spectroscopy HPLC-AAS has recently seen several new or improved interfaces. Thermospray has been used for the determination of metals in complex matrices [227,228], and thermochemical hydride generation has been used for arsenic determinations [229]. A thermo interface for the determination of butyltin in wood preservatives [230], a hydraulic high-pressure nebulizer for the determination of Cu, Fe, Ni, Cd, and Mg [231], and a glass capillary array nebulizer [232] have also been described.

Parks et al. have shown an enhanced signal for organotin and organolead compounds using oxides of transition metals [233]. They suggested that the enhancement was due to the formation of relatively nonvolatile metal oxides. Li et al. [234] developed a method for separating and detecting noble metals, and Xia et al. [235] used HPLC-AAS to quantitate metallothioneins.

B. Flame Methods

Above, element-specific flame methods such as flame AAS and flame emission were mentioned. Here two nonelement specific methods will be briefly described. They are flame ionization and flame infrared emission.

1. Flame Ionization Flame ionization detectors (FIDs), also called transport detectors, have been described by Hinshaw [236]. Although his discussion is for gas chromatography applications, it gives an excellent description FIDs in general. The use of FIDs


for HPLC has been reviewed by Brown [237] and Vickrey and Stevenson [238]. FIDs respond to many types of hydrocarbons and have a large dynamic range.

HPLC-FIDs differ mainly from GC-FID in the interfacing. Normally HPLC-FID interfaces involve solvent removal, traditionally by moving belts or wires. These tend to be quite noisy and limit the use of buffers, ion pairing agents, and salts [239]. These detectors were popular in the 1970s with several commercial detectors available, all of which were eventually taken off the market. Recently, improvements have been described for HPLC-FID systems [240-242] and a commercial FID was introduced [243,244].

2. Flame Infrared Emission Flame infrared emission (FIRE) detection for HPLC is a new technique that is mainly used for organic acids in aqueous media [245,246]. It involves the combustion of organic molecules to carbon dioxide followed by detection at 4.3-4.4 and 2.7-2.9 microns [247,248]. More recently characteristic bands for HCl and HF have been identified that may prove useful for the analysis of halogenated hydrocarbons [249]. More developments using this technique will probably become available in the near future.

C. Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR), like IR, is an information-rich technique. It gives valuable information that can be used for structure elucidation and stereochemical determinations. Continuous flow studies have been done using both proton and carbon NMR. In addition, studies have been done comparing stop and continuous-flow detection [250,251]. Dorn has discussed HPLC-proton NMR in detail [252].

The main problem that exists in HPLC-NMR is solvent supression, particularly that of protonated solvents in reverse-phase systems. The techniques used for supression of solvent signals include solvent nonexcitation and selective excitation of a distinct NMR region. The former procedure was introduced by Hore [253,254] for the separation of aromatic compounds and later used by Albert et al. for cyclopropyl containing drugs [250], dansyl amino acids [255], and aromatic compounds [256]. Selective excitation of distinct NMR regions was introduced by Clore et al. [257] and used by Laude et al. for biomolecules [258] and three different mixtures: phenols, vitamins, and analgesics [259].

An alternative approach to solvent supression is to eliminate the need altogether by using nonprotonated (i.e., deuterated) solvents. This, however, is prohibitively expensive unless microbore columns are used.

A second, lesser problem in HPLC-NMR is obtaining good spectral characteristics such as line shape and signal-to-noise ratio. Albert et al. studied the effect of flow cell volume on peak broadening [255] and showed improvements in line widths and sensitivities using a flow cell with a special detector coil [256]. Haw et al. briefly studied the line width contribution from residence time for flow rates of 2.5 to 5 mL/min and found the effect was minimal [260].


D. Multidimensional Chromatography

When there is a need to separate complex mixtures into individual components, often one Chromatograph is not sufficient. In these cases, two or more chromatographs may be used together in series. The first (here the liquid Chromatograph) separates the sample into classes, and the second separates the classes into individual components [261-263]. These techniques are of interest here because they allow detection by methods not normally readily accessible to HPLC eluates. The chromatographs most often interfaced with liquid chromatographs (besides other liquid chromatographs) are gas chromatographs. HPLC has also been interfaced with thin-layer chromatography and capillary-zone electrophoresis [264].

1. HPLC-GC HPLC coupled to GC is finding applications in the analysis of petroleum fuels [261,265-267], foodstuffs [267,268], coal-derived fuels [267,269,270], environmental samples [262,267,271], medical samples [267] and metal chelates [272]. It has been reviewed by several authors [267,268,273-275] and a commercial system is now available (266,271,276).

Interfacing an HPLC to a GC consists of three basic steps. The peak of interest must first be found; the solvent volume is then reduced and an injection is made into the GC. Choosing the LC peak or peaks of interest is usually done using a UV detector.

Solvent volume reduction is necessary because the mobile phases of LC and GC differ in that one is a liquid and the other a gas. LC solvents can interfere in GC methods and therefore must be carefully chosen and reduced prior to GC introduction. The reduction may be done by stream splitting [269,270], which reduces sensitivity, LC miniaturization [277, 278], or solvent evaporation using a retention gap to avoid band broadening [265,267,279,280].

LC solvents should be carefully chosen; in general, normal-phase solvents work better than reverse-phase ones. The solvent must be compatible with both the LC and the GC detectors. It also must have a lower boiling point than the solutes and preferably a high vapor pressure for rapid removal. When reverse-phase solvents are present, a solvent exchange interface may be used [267].

2. HPLC-TLC

Both normal and reverse-phase HPLC can be interfaced with thin-layer chromatography (TLC) [281]. This involves attaching a capillary tube to the end of the HPLC column, dropping the eluate onto a TLC plate, developing the plate, and detecting the spots. Fujimoto et al. used IR detection [282-284]. Karmen et al. [18] used radioactivity detection with a solvent concentration step prior to deposition.

E. Other

Barth et al. [285] has reviewed other detectors used for HPLC. Some of these will be briefly discussed here.


1. Raman Spectroscopy Raman spectroscopy, like IR spectroscopy, gives molecular structure information. It has advantages over other techniques in that it is nondestructive and is free from water interference. Raman spectra of HPLC eluates have been obtained off-line [286], on-line [287,288], and using stopped-flow conditions [289,290]. Detection techniques include surface-enhanced Raman scattering [287,288,290], surface-enhanced resonance Raman scattering [286], and resonance Raman scattering [289]. On-line detection limits are reported as 175 pmol for adenine, 233 pmol for thymine, and 211 pmol for cytosine [287].

2. Dielectric Constant Dielectric constant (DC) detection of hydrocarbon distillates has been described [291,292] and reviewed [293]. A commercial DC detector has been evaluated [294] and newer designs have been discussed [295,296].

3. Viscometry Viscometer detectors have been used in combination with differential refractive index detectors in gel permeation chromatography in the analysis of exopolysac-charides [297] and to determine molecular weight data and branching information of polyolefins [298-301]. Viscometry detectors have been compared to osmometry and low-angle laser light-scattering (LALLS) [299,302] because they provide similar information. Recently, a commercial GPC/viscometry chromatography system was introduced [199].

4. Ultrasonic An ultrasonic detector has been developed for use in industrial-scale separations [303,304] with flow rates of 80 to 5000 mL/min. The detector is based on the principle that the ultrasonic frequency transmitted through a liquid varies according to the substance type and density. The detector was studied using polyethylene glycol standards.

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A Practical Guide to HPLC Detection || OTHER MODES OF DETECTION

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