## Summary of detectors used in HPLC

For the majority of analyses of drugs in formulations, variable wavelength UV or diode array UV detectors are used. A typical UV dector has a narrow cell about 1 mm in diameter with a length of 10 mm, giving it an internal volume of about 8 fA. The linear range of such detectors is between 0.0001 and 2 absorbance units and samples have to be diluted sufficiently to fall within the range. Although the exact concentration of a sample passing through the flow cell is not known, a suitable concentration can be approximated as shown in Calculation example 12.3.

### Calculation example 12.3

A typical elution volume of chromatographic peak volume is 400 ¡A. If 20 ¡A (0.02 ml) of a solution containing paracetamol at a concentration of I mg/100 ml is injected into an HPLC system with a flow cell with a pathlength of 10 mm:

Amount of paracetamol injected = 1 mg x 0.02/100 = 0.0002 mg.

Mean concentration of paracetamol in the peak volume = 0.0002 x 100/0.4 = 0.05 mg/100 ml.

The A(l%, 1 cm) value for paracetamol at 245 nm is 668.

The absorbance of a 0.05 mg (0.00005 g) solution = 0.00005 x 668 = 0.0334.

The mean absorption across the peak would be 0.00334.

If the peak has a Gaussian shape, the maximum absorption for the peak would be cci 1.5 times the mean absorption, i.e. in this case 0.05 or 50 milliabsorbance units (mAU).

Selective detectors tend to be employed where the analyte is present in small amounts in a complex matrix such as in bioanalytical procedures where components extracted from the biological matrix along with the analyte can cause interference. Some formulated compounds have only very poor chromophores - these include: sugars, lipids, surfactants, amino acids and some classes of drugs, e.g. a number of anticholinergic drugs lack chromophores. In these cases an alternative to UV detection has to be employed.

Table 12.2 Some detectors commonly used In HPLC

### Detector Applications

Based on absorption of UV light by an analyte. A robust detector with good sensitivity works approximately in the range of 0.01-100 /jg of a compound on-column. The sensitivity of the detector in part depends on the <4(1%, 1 cm) value of the compound being analysed. The early detectors operated at a fixed wavelength (usually 254 nm); currently detectors are available which can be adjusted to operate at any wavelength over the full UV/visible range

Detector

Variable wavelength UV detector

Variable wavelength UV detector

Detector

Table 12.2 Some detectors commonly used in HPLC (Cont.)

Detector

Applications

polychromatic

Dispersion o o O o o polychromatic

Flow

Dispersion o o O o o

Evaporative light scattering detector (ELSD)

Aerosol Detector

Aerosol Detector

Electrochemical detector

Flow

Ag/AgCI Reference

^^g—Flow Auxiliary

Flow

Carbon working electrode

Carbon working electrode

### Pulsed amperometric detector

An advanced type of UV detector with the ability to monitor across the full UV range simultaneously using an array of photodiodes which detect light dispersed by a fixed monochromator over a range of wavelengths offering a resolution of ca 1 nm. Useful for complex mixtures containing compounds with widely different absorbance ranges and for mixtures where peaks overlap chromatographically but can be separated in terms of UV absorbance. The detector gives a full UV spectrum of each peak in the chromatogram which aids in identification of unknowns

Detection is based on the scattering of a beam of light by particles of compound remaining after evaporation of the mobile phase. This detector is of growing importance; it is a universal detector and does not require a compound to have a chromophore for detection. Applications include the analysis of surfactants, lipids and sugars. Unlike the refractive index detector, which was formerly used for this analysis, it can be used with gradient elution and is robust enough to function under a wide range of operating conditions. However, it cannot be used with involatile materials such as buffers in the mobile phase or to detect very volatile analytes. Typical applications include: analysis of chloride and sodium ions in pharmaceuticals, lipids used as components in formulations, sugars and sugar polymers. Sensitive to ca 10 ng of analyte

The electrochemical detector is usually used in the coulometric mode. A fixed potential is applied between the working and reference electrode. Detection is based on production of electrons when the analyte is oxidised, which is the more common mode of operation, or consumption of electrons in the reductive mode. The current flowing across the detector cell between the working and auxiliary electrodes is measured. The working electrode that carries out the oxidation or reduction is usually made from carbon paste. Most applicable to selective bioanalyses such as the analysis of drugs in plasma, e.g. catechols such as adrenaline and thiol drugs such as the angiotensin-converting enzyme inhibitor captopril and the anti-rheumatic drug penicillamine

There is really no distinction between this detector and an electrochemical detector except that the detector has arisen largely as part of ion chromatography and tends to be used in the amperometric mode where conduction of current between two electrodes by an ionic analyte is measured rather than current changes resulting from oxidation or reduction of the analyte. The working electrode in this detector is usually gold rather than carbon paste. Highly sensitive to ionic compounds, the detector is used in ion chromatography for the analysis of inorganic ions such as phosphate and sulphate. Typical pharmaceutical applications include the analysis of cardenolides and aminoglycoside antibiotics which do not have chromophores. Sensitivity is typically down to 1 ng of analyte. Widely used in glycobiology for the analysis of sugar residues derived from glycoproteins. In the pulsed mode, the polarities of the electrodes are alternated in order to keep the electrode surfaces clean

Table 12.2 Some detectors commonly used in HPLC (Co/it.)

### Detector Applications

Detection is based on changes of refractive index when the analyte passes through the sample cell (Samp.) in the detector, the reference cell (Ref.) being filled with the mobile phase. Like the ELSD, the Rl detector is a univeral detector with even less selectivity than the ELSD. It is very sensitive to mobile phase composition and temperature making it non-robust. It is still used as a universal detector since it is cheaper than an ELSD. Sensitive to ca 1 ¡jg of compound

Detection is based on fluorescent emission following excitation of a fluorescent compound at an appropriate wavelength. A robust and selective detector applicable to compounds exhibiting fluorescence and to fluorescent derivatives. Most useful for selective bioanalyses. Sensitive to below the ng level for highly fluorescent compounds. Normally uses a Xenon lamp for excitation but instruments with high intensity deuterium lamps are available for excitation of short wavelength absorption bands

HKw Self-test 12.4

Rank the following detectors in order of decreasing: a. Selectivity b. Robustness c. Sensitivity:

(i) Variable wavelength UV detector.

(iii) Rl detector.

(iv) Electrochemical detector.

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