Introduction to Modern Liquid Chromatography, Third Edition part 21 pdf

Table 4.1Improvement of Signal-to-Noise Ratio Better wavelength or other detector adjustment Increase detector time constant Inject larger weight of sample Higher reagent/solvent purity

N S

Figure4.7 Measurement of chromatographic signal (S) and noise (N)

from the middle of the baseline noise to the top of the peak (Fig 4.7) The

contribution of S /N to precision can be estimated as

CV= 50

where CV is the coefficient of variation (equivalent to %-relative standard deviation,

%-RSD) The lower limit of detection (LLOD) often is described as S/N = 3,

which would give CV ≈16%, whereas the lower limit of quantification (LLOQ)

is S /N = 10, for CV ≈5% (see also Section 11.2.4) These values of CV are

the contribution of S /N to the overall imprecision of the method, so the overall

method precision is expected to be worse than the S /N contribution As long as the

imprecision attributable to S /N (or any other single contributor to error) is less than

half of the desired method imprecision, S /N will have a minor (<15%) influence on

the overall method precision (see the discussion of Eq 11.2) For example, if the

overall method requires imprecision of no more than 2%, a contribution of S /N of

<1% should be satisfactory This suggests that a S/N value of ratio 50:1 or more is

required for an overall method imprecision of<2%.

The signal-to-noise ratio can be improved by increasing the signal, reducing the noise, or both, as summarized in Table 4.1 An increase in signal for a given peak or sample may be available from a change in detector setting; for example, the use of a UV wavelength that corresponds to maximum sample absorptivity A more sensitive detector also may be available, of either the same or different type Derivatization (Section 16.12) or other modification of the analyte may make

it more responsive to the chosen detector A more common means of increasing the signal is to inject a larger weight of sample (either a larger sample volume or

a sample concentrate; Section 3.6.3) However, column or detector overload will eventually limit the possible increase in signal in this way Any reduction in peak width should translate into a proportional increase in peak height (area is assumed

to be constant); smaller k-values (increase in %B; see examples of Fig 2.10b),

narrow-diameter columns, or more efficient small-particle columns can each be used for this purpose

Table 4.1

Improvement of Signal-to-Noise Ratio

Better wavelength (or other detector adjustment) Increase detector time constant

Inject larger weight of sample Higher reagent/solvent purity

Larger plate number

Any reduction of baseline noise also can improve S /N, for example, signal

smoothing by an increase in the detector time-constant or data-system sam-pling rate (Section 4.2.3.1) Excessive smoothing, however, can reduce the signal intensity Better temperature control of the column, detector, and general instru-ment environinstru-ment also can reduce noise, especially for detectors sensitive to refractive index changes Purer solvents (e.g., HPLC grade) and better sam-ple cleanup can reduce the introduction of noise-generating contaminants For gradient applications, changes in the system are sometimes attempted in order

to reduce the dwell-volume (Section 9.2.2.4) and the gradient delay time The mixer-volume comprises a major fraction of the dwell-volume in many systems, but reduction of the mixer-volume can increase baseline noise Some HPLC sys-tems have optional mixers that can be added to smooth the baseline and reduce noise—these devices can be especially advantageous for isocratic methods run

at maximum detector sensitivity Column switching (Section 16.9) can be used

to transfer a desired fraction from a cleanup column to the analytical column, thereby diverting unwanted contaminants to waste, so as to reduce baseline noise

4.2.4 Detection Limits

Although the signal-to-noise ratio is a measure of the inherent quality of the detector signal, the minimum detectable mass or concentration often is the limiting factor

in the usefulness of a detector for a particular application The term sensitivity often is used interchangeably with detection limit when describing an HPLC

detec-tor However, in proper use, sensitivity is the slope of a calibration plot, that

is, the change in signal per unit change in concentration (or mass) of analyte, whereas detection limit refers to the minimum concentration (or mass) that can

be measured HPLC detectors respond either to the concentration of the sample in the detector cell (e.g., UV detection) or the mass of sample in the detector (e.g., LC-MS)

Detection limits, discussed more thoroughly in Section 11.2.5, are defined as

follows: The limit of detection LOD is the smallest signal that can be discerned

from the noise—with confidence that a peak really is present Often a S /N of 3 is

equated to the LOD The lower limit of quantification LLOQ (sometimes called

limit of quantification or limit of quantitation, LOQ) is the smallest signal that can

be measured with the required precision for the method The LLOQ often is defined

as S /N ≥ 10, but a value of S/N ≥ 50 may be chosen for high-precision methods.

There is a never-ending need for lower and lower detection limits for trace analysis, and assays for which on-column injections of<1 ng are becoming more and more

common

The LOD and LLOQ are directly related to the concentration (or mass) of

sample in the detector cell Thus a longer path-length cell for UV detection is favored

in terms of signal intensity However, the detector cell should be designed with

a minimum volume that is compatible with other requirements of the detector Excess cell volume will result in additional extra-column peak broadening (Section 3.9) This is especially true for small-volume columns, columns packed with small

particles, and peaks with k <2 For example, with a 50 × 4.6-mm column packed

with 3-μm particles and k<3, significant peak broadening was observed for an 8-μL UV-detector cell when compared with a 1-μL cell [14] To minimize the broadening

of early-eluted peaks, the detector cell volume V detshould be less than approximately

one-tenth of the final volume of the peak of interest V p (V p = WF, where W is the baseline width of the peak [min], and F is flow rate [mL/min]) [15]:

(For other peak-broadening contributions to V p, see Eq 3.1 in Section 3.9.)

Some examples of the column contribution to peak volume V p0for early-eluted

peaks (k= 2) for some popular column configurations are shown in Table 4.2 (In a well-behaved system, according to Eq 2.27 and 3.1, the observed peak

volume V p should not be much larger than V p0.) Table 4.3 lists the detector cell volumes for several UV-detector configurations For UV detectors (Section 4.4), signal intensity is proportional to path length, so longer path flow cells will have lower detection limits However, for detector cell diameters<1 mm, signal loss due

to light scattering in the cell can be a problem, so special cell designs (e.g., total internal reflectance) are necessary for smaller cell diameters (see the discussion of

Section 4.4) The data of Tables 4.2 and 4.3 show that column lengths L≥ 100

mm with a diameter d c = 4.6 mm, packed with 5- or 3-μm d p particles, will work well with the standard 10× 1.0-mm UV cell (see (Eq 4.2), but any combination of

smaller column dimensions or smaller particles requires smaller cell volumes to avoid unnecessary extra-column peak broadening (Note that Eq 3.1 is an approximation,

so peak-broadening calculations based on Eq 3.1, and therefore conclusions based

on Tables 4.2 and 4.3, also are approximations.)

4.2.5 Linearity

For quantitative analysis by HPLC (Section 11.4), the detector response must

be related to the amount of analyte present If analyte response y is plotted against analyte concentration x, the simplest, most convenient, and most reliable relationship is y = mx, where the slope m is a constant defined as the sensitivity Such a relationship between analyte response and analyte amount is termed linear.

Table 4.2

Typical Peak Volumes V p0

Table 4.3

UV-Detector Cell Volumes

For best use over a wide range of sample concentrations, a wide linear dynamic range

(the concentration range over which the detector output is proportional to analyte concentration, e.g., 105 for UV detection) is desired, so that both major and trace components can be determined in a single analysis over a wide concentration range For example, with a stability-indicating method, peaks≥ 0.1% of the response of

the active ingredient (= 100%) must be reported, which would require a linear range

of at least 100/0.1 = 103 Some detectors (e.g., evaporative light scattering) have a narrow linear range of 1 to 2 orders of magnitude Although less convenient and reliable, a nonlinear calibration curve (e.g., quadratic) can be used—as long as the detector response changes in a predictable manner with sample concentration (or mass)

Table 4.4

HPLC Detectors

Sample-Specific

(Sections 4.4–4.10)

Bulk Property (Sections 4.11–4.13)

Hyphenated (Sections 4.14–4.15)

Reaction (Section 4.16)

Electrochemical Corona discharge Nuclear magnetic

resonance Radioactivity

Conductivity

Chemiluminescent

nitrogen

Chiral

The remainder of this chapter (Sections 4.4–4.16) provides a discussion of most HPLC detectors in use today In Table 4.4, detectors are grouped by technique (sample specific, bulk property, etc.) in approximate order of popularity within each group Sample-specific detectors will be treated first, and reaction detectors last—with only limited discussion of less-used detectors Within each section, principles of detector operation are discussed first, followed by one or more example applications Where appropriate, a comparison with other detectors is included

A detailed discussion of every detector is beyond the scope of this book In addition to the references cited in each section, a more general discussion of HPLC detectors can be found in [16, 17]

The most widely used detectors in modern HPLC are photometers based on ultravio-let (UV) and visible light absorption These detectors have a high sensitivity for many solutes, but samples must absorb in the UV (or visible) region (e.g., 190–600 nm) Sample concentration in the flow cell is related to the fraction of light transmitted

through the cell by Beer’s law:

log



I o I



where I o is the incident light intensity, I is the intensity of the transmitted light, ε is

the molar absorptivity (or molar extinction coefficient) of the sample, b is the cell path-length in cm, and c is the sample concentration in moles/L Light-absorption

HPLC detectors usually are designed to provide an output in absorbance that is

linearly proportional to sample concentration in the flow cell,

A= log



I o I



where A is the absorbance.

Properly designed UV detectors are relatively insensitive to flow and temper-ature changes UV photometers that are linear to >2 absorbance units full scale

(AUFS) with <1 × 10−5 AU noise are commercially available With this perfor-mance, solutes with relatively low absorptivities can be monitored by UV, and it is possible to detect a few nanograms of a solute having only moderate UV absorbance The wide linear range of UV detectors (≈105) makes it possible to measure both trace and major components in the same chromatogram

UV detectors commonly use flow cells of the Z-path design of Figure 4.3a,

with a 1-mm diameter and 10-mm path length (for a volume of≈8 μL) This cell volume is adequate for≥100 × ≥4.6-mm columns packed with ≥3-μm particles

(Section 4.2.4), but smaller volume and/or smaller particle columns may experience unacceptable extra-column peak broadening in an 8-μL cell Shorter path-length cells will reduce the cell volume, but the signal is proportional to the path length (Eq 4.4)—so sensitivity must be balanced against extra-column peak broadening in choosing the flow cell dimensions If the refractive index (RI) within the cell changes (e.g., during gradient elution), the amount of energy reaching the photodetector can change; when a light ray hits the side of the flow cell, the ratio of reflected to absorbed light depends on the refractive-index ratio of the mobile phase and cell wall (and the angle of the light ray hitting the cell wall) The latter refractive-index effect plus imperfections in optical alignment make it difficult to successfully use cell diameters smaller than≈1 mm One innovation that can minimize this problem is a flow cell

design as in Figure 4.3b, where the internal surface of the flow cell is coated with a

reflective coating—light that strikes the sides of the flow cell is reflected so as to still reach the photodetector [18, 19] The use of this light-pipe technique allows the cell diameter to be reduced for smaller cell volumes (e.g., 0.25 mm × 10 mm ≈0.5 μL),

and thus less peak spreading for use with very small-volume, small-particle columns

Alternatively, a longer, narrower diameter flow cell can be used (increasing b in Eqs.

4.3 and 4.4) for more absorbance in a smaller volume cell (e.g., 0.25 mm i.d × 50

mm long, with a volume of≈2.5 μL).

It is not necessary to operate a UV detector at the absorption maximum of the analyte A hypothetical example of wavelength selection is shown in Figure 4.8 The

spectra for two analytes, X and Y, are shown in Figure 4.8a, with UV maxima at

≈250 nm and ≈270 nm, respectively At 280 nm, Y has much stronger absorbance,

so it has a much larger peak (Fig 4.8b, same mass on column) At 260 nm, the absorbances of X and Y are approximately equal, so the peaks are of approximately equal size (Fig 4.8c) At 210 nm, both compounds have even stronger absorbance and generate much larger peaks (Fig 4.8d) Notice also the appearance of a new peak Z, which was not observed at higher wavelengths This general increase in

sensitivity at lower wavelengths is one reason for the widespread use of≤220 nm for

detection (near-universal detection) The corresponding loss of detector selectivity

at lower wavelengths can be a disadvantage for other separations, where it might

0 1 2 3 4 5

Time (min)

280 nm

260 nm

X Y

Z

Wavelength (nm)

(a)

(b)

(c)

(d )

X

Y

Figure4.8 Wavelength selectivity for UV detection (a) Absorbance spectra for two hypo-thetical compounds X and Y Chromatograms at (b) 280 nm, (c) 260 nm, and (d) 210 nm.

be undesirable to ‘‘see’’ certain sample constituents (e.g., arising from the sample matrix)

UV-visible spectrophotometric detectors can respond throughout a wide wave-length range (e.g., 190–600 nm), which enables the detection of a broad spectrum

of compound types Almost all aromatic compounds absorb strongly below 260 nm;

lamp filter beam

splitter

sample &

reference flow cells

photocells

Figure4.9 Schematic of a fixed-wavelength UV detector Dashed lines show optical path

compounds with one or more double bonds (e.g., carbonyls, olefins) can be detected

at wavelengths of <215 nm, while the preponderance of aliphatic compounds

possess significant absorbance at≤205 nm Reversed-phase mobile phases of ace-tonitrile plus water or phosphate buffer can be used routinely for detection at

200 nm, whereas methanol-containing mobile phases cannot be used below≈210

to 220 nm, depending on methanol concentration (see Appendix I, Table I.2) The proper selection of the mobile phase makes it possible to operate UV detectors in

a near-universal detection mode in the 200- to 215-nm region, where most organic compounds exhibit some UV absorbance Because of the relatively small absorbance differential between water (or phosphate buffer) and acetonitrile at >200 nm or

methanol at >220 nm, UV detectors are also quite useful for gradient elution.

Mobile phases with large differences in UV absorbance, such as tetrahydrofuran and water at<240 nm, may not be amenable for use with gradients and UV detection.

UV detectors come in three common configurations Fixed-wavelength

detec-tors (Section 4.4.1) rely on distinct wavelengths of light generated from the lamp,

whereas variable-wavelength (Section 4.4.2) and diode-array (Section 4.4.3)

detec-tors select one or more wavelengths generated from a broad-spectrum lamp

4.4.1 Fixed-Wavelength Detectors

Figure 4.9 is a generic schematic of a fixed-wavelength UV detector These detectors were the mainstay of UV detection prior to the introduction of the variable- and diode-array detectors, but they are not widely used today Their current appeal is low price and simple construction, and they tend to be more popular in the educational environment or other budget-limited settings

UV radiation at 254 nm from a low-pressure mercury lamp passes through a band-pass filter and beam splitter, and shines on the entrance of the flow cell Light transmitted through the flow cell strikes the photodetector (usually a photodiode) and is converted to an electronic signal Most UV detectors operate in a differential absorbance mode, where light also passes through a reference cell, and the difference between the light passing through the sample and reference cells is converted

to absorbance, according to Equation (4.4) Although some detectors enable the reference cell to be filled with mobile phase, an air reference is most commonly used, which allows for correction of variations in light intensity from the source lamp, but not for changes in the mobile-phase absorbance

diffraction grating

slit

flow cell photocell

200 nm

360 nm

Figure4.10 Schematic of a variable-wavelength UV detector; reference flow cell not shown Dashed lines show optical path

The 254-nm line from the low-pressure mercury lamp is the most popular wavelength for use with the fixed-wavelength UV detector For historical reasons this wavelength is still popular for applications that use variable- and diode-array detectors, although there is no real reason to use this particular wavelength Through the use of other lamps (e.g., zinc), phosphors, and other lines in the mercury lamp output, detection at 214, 220, 280, 313, 334, and 365 nm can be accomplished with the fixed-wavelength detector

4.4.2 Variable-Wavelength Detectors

UV spectrophotometers (variable-wavelength and diode-array detectors) offer a wide selection of UV and visible wavelengths Such devices have the versatility and convenience of operation at the absorbance maximum of a solute or at a wavelength that provides maximum selectivity, as well as the ability to change wavelengths during a chromatographic run

The most widely used detector in HPLC today is the variable-wavelength UV detector shown schematically in Figure 4.10 A broad-spectrum UV lamp (typically deuterium) is directed through a slit and onto a diffraction grating The grating spreads the light out into its component wavelengths, and the grating is then rotated

to direct a single wavelength (or narrow range of wavelengths) of light through the slit and detector cell and onto a photodetector These detectors usually use a sample and reference cell configuration (Section 4.4) for differential detection For detection

in the visible region, a tungsten lamp is used instead of deuterium

The use of a variable-wavelength detector allows one to program a change in the detection wavelength during a chromatogram Thus one peak can be detected at

280 nm and another at 220 nm Although it is possible for many detector models

to change the wavelength quickly, so as to generate a UV spectrum for a peak, the results are complicated by the change in analyte concentration during the spectral scan and may be of limited value

slit

flow cell

photodiode array

diffraction grating

200 nm

360 nm

Figure4.11 Schematic of a diode-array UV detector Dashed lines show optical path

4.4.3 Diode-Array Detectors

A schematic of the diode-array detector (DAD, also called photodiode-array, PDA)

is shown in Figure 4.11; it has a similar optical path to the variable-wavelength detector, except that the white light from the lamp passes through the flow cell prior

to striking the diffraction grating This allows the grating to spread the spectrum

across an array of photodiodes, hence the name photodiode array (PDA) The number

of photodiodes varies with the specific brand and model of detector, but detectors with 512 or 1024 diodes are common The signals from the individual photodiodes are processed to generate a spectrum of the analyte Because the spectra are generated

at the same time (vs single-wavelength monitoring with the variable-wavelength detector), the DAD can contribute to peak identification The DAD can be operated

to collect data at one or more wavelengths across a chromatogram, or to collect full spectra on one or more analytes in a run Of course, the data-file size is much larger for full-spectra runs, but data compression techniques and inexpensive data storage make this less of a concern than it was in the past

If two closely eluted peaks have sufficiently different spectra, it may be possible

to distinguish the two peaks spectrally The utility of the DAD to distinguish between two peaks can be understood in conjunction with Figure 4.12, where a

partial chromatogram for a closely eluted peak pair X and Y is shown (Fig 4.12a).

If the solutes have spectra as shown in Figure 4.12b and are monitored at a

wavelength where both have significant absorbance, such as 260 nm, the resulting

chromatogram will look like a single peak (X + Y in Fig 4.12a); the corresponding

peaks are shown for the solutes injected individually Even though the peaks appear

to overlap completely at 260 nm, if other wavelengths are monitored, it may be

possible to distinguish between the peaks For example, if 240 nm is used, only X will respond, whereas only Y will respond at 280 nm The added selectivity of the

detector can be used to compensate, at least in part, for inadequate chromatographic separation Thus the DAD could simultaneously collect data at 240 and 280 nm during the chromatographic run, and individual chromatograms plotted at 240 or

280 nm would allow quantification of X and Y, even under the partially overlapped

conditions of Figure 4.12

Another common application of the DAD is for peak-purity determination The software accompanying the DAD accomplishes this by calculating an absorbance