Introduction to Modern Liquid Chromatography, Third Edition part 22 potx

UV-Detector Characteristics Capable of very high sensitivity for samples that absorb in the UV Good linear range >105 Can be made with small cell volumes to minimize extra-column band b

210 220 230 240 250 260 270 280

Wavelength (nm)

(b)

X

Y

X + Y (260 nm) (a)

Figure4.12 Illustration of spectral deconvolution of analytes (a) Hypothetical chro-matograms for individual injections of X and Y at 260 nm shown with combined response

X + Y at 260 nm; (b) spectra for X and Y.

ratio across the peak The same dataset collected at 240 and 280 nm could be used to determine peak purity by calculation of the 240/280 ratio at every point

across the peak If the peak were pure X or Y, the ratio would be constant, whereas if the mixture of Figure 4.12a were present, the ratio would be >1 when

nature of the ratio would indicate the presence of a peak mixture, even though the peaks overlapped chromatographically and appeared as a single peak at 260 nm Peak-purity algorithms compare the consistency of the spectrum across the entire peak and in some cases can identify the presence of minor impurities (e.g.,<1%)

that are eluted under the tail of the major peak For additional examples of the determination of peak purity by DAD, see [20, 21] or the detector manufacturer’s literature (e.g., [22])

4.4.4 General UV-Detector Characteristics

Table 4.5 summarizes the general characteristics of UV detectors UV detectors are ideal for use with gradient elution; many common, UV-transmitting solvents are available in HPLC grade for use as mobile phases (Tables I.2 and I.3 of Appendix I) The UV detector is very useful for the trace analysis of UV-absorbing solutes, but its widely varying response for different solutes can be a disadvantage if the compound

of interest does not absorb in the UV (or visible) region UV detectors are reliable and easy to operate, and are particularly suitable for use by less-skilled operators

UV-Detector Characteristics

Capable of very high sensitivity (for samples that absorb in the UV)

Good linear range (>105 )

Can be made with small cell volumes to minimize extra-column band broadening

Relatively insensitive to mobile-phase flow and temperature changes

Very reliable

Easy to operate

Nondestructive of sample

Widely varying response for different solutes

Compatible with gradient elution

Detection wavelength can be selected

Internal troubleshooting and calibration checks are common

Built-in test procedures that can be carried out at detector startup identify many potential detector problems and can provide automatic wavelength calibration The background, or baseline absorbance, of UV detectors can increase with continued use This usually indicates that the cell windows have become dirty and need cleaning or replacement Regular detector-cell flushing (as when the column is flushed) and sample cleanup can make more thorough cell cleaning a rarity Lamp life, a concern in the past, is seldom an issue today Useful lifetimes of>2000 hr are

common, and internal circuitry monitors lamp performance and can alert the user when the lamp output has deteriorated Although the linear response range of UV detectors may be>2 AU, according to manufacturer’s specifications, most analysts

try to operate the detectors at <1 AU for best results Stabilizing the flow-cell

temperature through thermostatting or use of a capillary-tubing heat exchanger helps to reduce noise and drift from flow rate or temperature changes

Figure 4.13a shows an example chromatogram for the determination of

derivatized roxithromycin (ROX) in human plasma by UV detection at 220 nm [23]

An internal standard, erythromycin (IS), was added to 50μL of plasma followed

by solid-phase-extraction sample cleanup and derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) With UV detection at 220 nm, the method could monitor plasma concentrations of ROX but was unable to reach the LLOQ of<1 μg/mL

necessary for pharmacokinetic studies (See discussion of Section 4.5 for comparison

of the UV response of Fig 4.13a for this sample to the fluorescence response of Fig 4.13b.)

4.5 FLUORESCENCE DETECTORS

Fluorescence detectors are very sensitive and selective for solutes that fluoresce when excited by UV radiation Sample components that do not fluoresce do not produce

a detector signal, so sample cleanup may be simplified For example, a simple acetonitrile/buffer extraction allowed detection of as little as 30 pg of (naturally

Time (min)

IS (area = 951)

ROX (area = 1901)

IS (area = 961)

ROX (area = 609)

(a)

(b)

UV

fluorescence

5

5

Figure4.13 Chromatogram for the determination of roxithromycin (ROX) in human plasma

by (a) UV detection at 220 nm, and (b) fluorescence detection (excitation 255 nm,

emis-sion 315 nm) Retention: ROX (10.7 min), internal standard erythromycin (5.1 min), both cleaned up by solid-phase extraction and derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl) Adapted from data of [23]

fluorescing) riboflavin in food products by HPLC with fluorescence detection [24] Fluorescent derivatives of many nonfluorescing analytes can also be prepared (e.g., [25]), and this approach can be attractive for the selective detection of compounds for which sensitive or selective detection methods are otherwise not available

A schematic of a fluorescence detector is shown in Figure 4.14 The light source usually is a broad-spectrum UV lamp, such as the deuterium lamp used in

UV detectors, or a xenon flash lamp The excitation wavelength is selected by a filter

or monochromator, and it illuminates the sample as it passes through the flow cell When a compound fluoresces, the desired emission wavelength is isolated with a filter or monochromator and directed to a photodetector, where it is monitored and converted to an electronic signal for data processing Because fluorescence is emitted

in all directions, it is common to monitor the emitted light at right angles to the incident light—this simplifies the optics and reduces background noise The least

filter or

sample out

Figure4.14 Schematic of a fluorescence detector Dashed lines show optical path

expensive fluorometers use filters to select both excitation and emission wavelengths, whereas the most expensive use two monochromators (allowing a wide choice for both excitation and emission wavelengths) Remember, the fluorescence process is not 100% efficient, so energy is lost This means that the emission wavelength always must be at lower energy (higher wavelength) than the excitation wavelength For many samples, the fluorescence detector is 100-fold more sensitive than

UV absorption—and is one of the most sensitive HPLC detectors In other cases the sensitivity advantage of fluorescence over UV detection may be smaller but adequate for the task at hand A comparison of the detector response to roxithromycin (ROX)

by fluorescence and UV is shown in the RPC separations of Figure 4.13 [23] ROX does not fluoresce naturally, so derivatization (9-fluorenylmethyl chloroformate [FMOC-Cl]) of the sample and internal standard (IS) was used to enable detection

by fluorescence When comparing the UV response (Fig 4.13a) to fluorescence (Fig 4.13b), the fluorescence response for the derivatized IS is approximately the

same as the UV response, but the derivatized ROX peak response tripled with fluorescence detection The baseline noise was approximately the same for both UV and fluorescence This increase in response by the fluorescence method was adequate

to reduce the LLOQ to<1 μg/mL of ROX in human plasma, which was required

for pharmacokinetic studies

Because of its high sensitivity the fluorescence detector is particularly useful for trace analysis, or when either the sample size is small or the solute concentration

is extremely low The linear dynamic range of the fluorescence detector usually is smaller than that of UV detectors, but it is more than adequate for most trace analysis applications While the dynamic range (the range over which a change

in sample concentration produces a change in the detector output) of fluorescence detectors can be fairly large (e.g., 104), the linear dynamic range may be restricted

for certain solutes to relatively narrow concentration ranges (as low as 10-fold) For all quantitative analyses using the fluorescence detector (or any other detector, for that matter), the linear range should be determined through the use of appropriate calibration (Section 11.4.1)

In comparison to other detection techniques, fluorescence generally offers greater sensitivity and fewer problems with instrument instability (e.g., from temper-ature and flow changes) If solvents and mobile-phase additives free of fluorescing materials are used, the fluorescence detector can be used with gradient elution The major disadvantage of the fluorescence detector is that not all compounds fluoresce

0 Time (min)

Figure4.15 Fluorescence quenching of naphthalene by dissolved oxygen in the mobile phase Mobile phase sparged with helium (He) or air, as shown Adapted from data of [25]

As with other fluorescence techniques, fluorescence detection can be compromised

by background fluorescence of the mobile phase or sample matrix, and by quenching effects An example of fluorescence quenching is shown in Figure 4.15 [25] When the mobile phase is sparged with helium, a consistent signal is observed, but when air is bubbled through the mobile phase, the signal drops because oxygen quenches the fluorescence of the naphthalene peak (250-nm excitation, 340-nm emission) Sparging the oxygenated mobile phase with helium then displaces the oxygen and the signal returns to normal The presence of oxygen in the mobile phase also shifts the baseline slightly, but this is of minor concern

The use of a laser (laser-induced fluorescence, LIF) as the excitation source is available in the LIF detector The higher energy of the laser over the conventional deuterium or xenon lamp gives added sensitivity to this detector, but the excitation wavelength range is more limited (300–700 nm vs 200–700 nm for conventional fluorescence) LIF detection is not widely used with conventional HPLC systems, but is more common with micro applications (micro-LC, capillary LC, capillary electrophoresis, etc.) where a small diameter (e.g., 100-μm i.d.) flow cell is required

to limit dispersion

4.6 ELECTROCHEMICAL (AMPEROMETRIC) DETECTORS

Many compounds that can be oxidized or reduced in the presence of an electric potential can be detected at very low concentrations by selective electrochemical (EC) measurements By this approach the current between polarizable and reference electrodes is measured as a function of applied voltage Because a constant voltage normally is imposed between the electrodes, and only the current varies as a result

of solute reaction, EC detectors are more accurately termed amperometric devices.

EC detectors can be made sensitive to a relatively wide variety of compound types, as illustrated in Table 4.6 EC detection is common for the determination of catecholamine and other neurotransmitters Many of the compounds in Table 4.6 also can be detected by UV absorption, but some compound types (e.g., aliphatic mercaptans, hydroperoxides) sensed by EC detection cannot be detected at all by

UV absorption, or only with difficulty and low sensitivity at low wavelengths

Table 4.6

Some Compound Types Sensed by the EC Detector

Aromatic halogens Nitro compounds Heterocyclic rings Note: Compound types generally not sensed include ethers, aliphatic hydrocarbons, alcohols, and car-boxylic acids.

aDetected depending on structure.

EC detectors can be used only under the condition that the mobile phase is electrically conductive, but this is a minor limitation, since most HPLC separations are done by reversed-phase with water or buffer in the mobile phase By fine-tuning the detector potential, one can achieve great selectivity for electroactive compounds The EC detector’s sensitivity makes it one of the most sensitive of all HPLC detectors, for example with detection limits to 50 fg on-column of dopamine However, to operate under high sensitivity, extra care must be taken to use highly purified mobile phases to reduce background noise In order to reduce the background noise, in some applications the mobile phase is routed through a high-potential pretreatment cell so as to oxidize or reduce background interferences before the mobile phase reaches the autosampler

A glassy carbon electrode is most commonly used in the electrochemical cell In the configuration shown in Figure 4.16, the column effluent flows across

a glassy carbon electrode, whereas in another popular configuration, the sample flows through a porous graphite electrode Several electrode styles are available, for

example, Figure 4.16c shows a dual-electrode configuration The high susceptibility

of the EC detector to background noise and electrode contamination has earned it a reputation as a difficult detector to use However, newer units are much more trouble free and can provide excellent and reliable results in the hands of a reasonably careful operator

Figure 4.17 shows the electrochemical detection of acteoside, an active ingre-dient in many Chinese medicinal plants Following intravenous administration of acteoside at 10 mg/kg, the analyte was detected in rat brain microdialysate at a concentration of≈25 ng/mL (≈0.4 ng on-column) by reversed-phase HPLC [26].

More information about electrochemical detectors can be found in [27]

sample inlet sample

outlet

locking

collar

reference electrode o-ring

auxiliary electrode block

gasket

working

electrode

quick-release mechanism

(a)

(b)

(c)

electrode flow

in

flow out

Figure4.16 Schematic of an electrochemical detector (a) Top view of assembled flow cell; (b) exploded diagram of cell; (c) detail of dual electrode cell Courtesy of Bioanalytical

Sys-tems, Inc

4.7 RADIOACTIVITY DETECTORS

Radioactivity detectors are used to monitor radio-labeled solutes as they elute from the HPLC column Detection is based on the emission of light in the flow cell

as a result of radioactive decay of the solute, followed by emission of α-, β-, or

γ -radiation The continuous-flow monitoring of β-radiation in the eluent ordinarily

involves the use of a scintillation technique, where the original radiation is converted

to light Depending on the method of combining the eluent and the scintillator, this

can be classified as either a homogeneous or heterogeneous system In homogeneous

operation, a liquid-scintillation cocktail is mixed with the column effluent prior to

entering the detection cell, where emitted light is monitored Under heterogeneous

conditions, the column outlet is routed directly into the detector cell, which is packed with beads of a solid scintillant When adsorption of the analyte on the beads is a problem, the scintillant may be coated onto the walls of the detector cell

Homogeneous detectors are best used with analytical procedures where recov-ery of the sample is unimportant The technique also can be applied to preparative HPLC, when a portion of the sample stream is split off to the detector Hetero-geneous detectors are less sensitive, and therefore better suited for samples with

0 5 10 15 20 25 0

0.4 0.8

Time (min)

acteoside

Figure4.17 Determination of acteoside (t R≈ 15 min) in rat brain microdialysate with elec-trochemical detection Adapted from data of [26]

higher levels of radioactivity (or for larger solute concentrations, as in preparative separations) Heterogeneous systems also are relatively free of chemical quenching effects, and solutes can be recovered easily However, this detector exhibits relatively low counting efficiency for low-energyβ-emitters, such as35S,14C,3H, and32P, and

is better suited for strongerα-, β-, and γ -emitters (e.g.,131I,210Po, and125Sb) One application of the radioactivity monitor is to determine the complete distribution and mass balance of a radio-labeled pharmaceutical dosed in an experimental animal Such determinations are difficult, if not impossible, without the aid of radio-labeled drugs

Radiochemical detectors have a wide response range and are insensitive to solvent change, making them useful with gradient elution With radioactivity detec-tors, it may be necessary to compromise sensitivity to improve chromatographic resolution and speed of analysis Detection sensitivity is proportional to the number

of radioactive decays that are detected, and this number is proportional to the volume of the flow cell and inversely proportional to the flow rate (proportional to residence time, which allows more atoms to decay during passage of a peak through the flow cell) Larger flow-cell volumes increase extra-column peak broadening and can diminish resolution, while slower flow rates mean an increase in separation time Because detection sensitivity is often marginal, larger flow cells are generally preferred for radioactivity detection

In practice, peak tailing and peak broadening in a radiometric flow cell can

be minimized by working with columns of larger volume (assuming that sufficient sample is available for larger mass injections to compensate for sample dilution).With radioactivity detection, a compromise between chromatographic resolution and detector sensitivity must be reached, the exact nature of which depends on the analytical requirements

4.8 CONDUCTIVITY DETECTORS

Conductivity detectors use low-volume detector cells to measure a change in the conductivity of the column effluent as it passes through the cell Conductivity detectors are most popular for ion chromatography and ion exchange applications

in which the analyte does not have a UV chromophore Analysis of inorganic ions (e.g., lithium, sodium, ammonium, potassium) in water samples, plating baths, power plant cooling fluids, and the like, is an ideal use of the conductivity detector Organic acids, such as acetate, formate, and citrate are also conveniently detected

by conductivity

Conductivity detection can be compromised by the presence of a conductive mobile phase; for example, the mobile-phase buffer Thus the presence of the buffer greatly increases the conductance of the mobile phase, which is only slightly increased by the presence of the solute One way to minimize this problem is to use

a suitable buffer in combination with a suppressor column (ion exchanger), in order

to reduce the background conductivity of the mobile phase For example, consider the need to detect one or more anionic solutes The use of a Na2CO3-NaHCO3

buffer with a cation-exchange suppressor column (termed an anion suppressor in

ion chromatography terms) in the H+ form will eliminate Na+ and other cations from the mobile phase, and convert carbonate to weakly acidic carbonic acid This reduces the conductivity of the mobile phase and allows an easier detection or small concentrations of anionic solutes The application of a suppressor column is illustrated in Figure 4.18 for the dramatic improvement in conductivity detector response to F−, Cl−, and SO2−4

4.9 CHEMILUMINESCENT NITROGEN DETECTOR

One advantage that gas chromatography has over HPLC is the availability of several element-specific detectors, allowing selective detection of compounds containing nitrogen, sulfur, or phosphorus In the 1970s much effort was given to developing element-specific detectors for HPLC, but for the most part the results have been discouraging One exception is the chemiluminescent nitrogen detector (CLND), which was reported as early as 1975 [28] Several commercial implementations and refinements have resulted in today’s CLND

The HPLC column effluent is nebulized with oxygen and a carrier gas of argon

or helium and pyrolyzed at 1050◦C Nitrogen-containing compounds (except N2) are oxidized to nitric oxide (NO), which is then mixed with ozone to form nitrogen dioxide in the excited state (NO2*) NO2* decays to the ground state releasing a photon, which is detected by a photometer The signal is directly proportional to the amount of nitrogen in the original sample, so calibrants of known nitrogen content can be used to quantify the nitrogen content of unknown analytes This is illustrated

in Figure 4.19a [29], where the injection of 50-ng nitrogen equivalents of 7 different

compounds give detector responses that are constant within ±10% Care must be taken to maintain a nitrogen-free mobile phase, so the use of acetonitrile is ruled

out Many solvents are compatible with the CLND, as is shown in Figure 4.19b for

the response of the injection of 1 mg each of 6 nitrogen-free solvents, compared to

an injection of 1 ng nitrogen-equivalent of a standard

F – Cl –

mobile phase

(Na2CO3)

sample (F – , Cl – , SO42– )

analytical

column

anion

suppressor

NaF, NaCl,

Na2SO4

in Na2CO3 waste

waste

HF, HCl, H2SO4

in H2CO3

Time

Time

Without Suppression

With Suppression counter ions

F –

Cl –

SO42–

SO42–

(c)

conductivity detector

Figure4.18 Use of an anion suppressor column to enhance conductivity detector response to

anionic analytes (a) Schematic of instrumentation; (b) conductivity detector output without suppressor column; (c) chromatogram with suppressor column in use Courtesy of Dionex.

One detector manufacturer claims detection limits equivalent to 0.1 ng of

nitrogen A practical example is seen in Figure 4.20a [30] for the detection of 13

underivatized amino acids by ion-pair chromatography and CLND The response per nitrogen atom is within 6% RSD, with detection limits of≈0.3 to 0.5 μg/mL for the amino acids Figure 4.20b shows the chromatogram for an injection of 10μL of wine filtered through a 1000-Da filter (note overloaded proline peak shows shorter

retention and strong tailing compared to a; see Section 2.6 for further discussion of

overload)

4.10 CHIRAL DETECTORS

Chiral drug candidates often are encountered in the development of new pharmaceu-tical compounds Different enantiomers can possess different efficacy, toxicology,

or other pharmacological characteristics, and the final product generally is a single enantiomer or a known mixture of enantiomeric forms Chromatographic separation

of the enantiomers (Chapter 14) is vital to the analysis of such mixtures Detection and identification can be further aided by the use of detectors that respond selectively

to specific chiral forms

Chiral detectors come in three different formats; each of these uses the same

principles as stand-alone instrumentation, but in a flow-cell format Polarimeters

(PL) measure the degree of rotation of polarized light (typically in the 400–700 nm range) as it passes through the sample The degree of rotation is dependent on