D e t e c t o r s f o r H P L C

1. D e t e c t o r s f o r H P L C

2. UV Detectors Fixed Wavelength Variable Wavelength Diode array

3. High Pressure Mercury Lamp Fixed Wavelength Mercury vapor lamps are probably the most common and emit intense light at 253.7 nm (and certain other wavelengths). Because of the limited emission spectra, of the lamp wavelengths are not adjustable. Because of the intensity of the radiation, fixed wavelength detectors can be up to 20 times more sensitive than variable wavelength detectors.

4. Variable Wavelength The variable types use a deuterium or similar lamp that produces a broad spectrum of wavelengths that are separated by a defraction grating. The grating works like a prism but generally the resolving power is higher for gratings than for prisms.

5. Diode Array The photo diode array detector passes a wide spectrum of light through the sample. The spectrum of light is directed to an array of photosensitive diodes. Each diode can measure a different wavelength which allows for the monitoring of many wavelengths at once. - Peak Purity - Quantify a peak with an interfering peak Compound Identification Monitor compounds with different UV max.

6. Intensity of Lamp Energy

7. Lamp Life As the lamps age, the light intensity drops significantly. Therefore it is important to monitor lamp usage. Arc lamps have an average lifetime of about 200 hours and most detectors are equipped with a timer that keeps track of lamp usage.

8. Fluorescence Detectors Fluorescence detectors are among the most sensitive of HPLC detectors, and depending on the compound, can be from 10-1000 times more sensitive than UV detectors analyzing strong UV absorbing compounds. The fluorescence detector is based on the principle that some compounds fluoresce when bombarded with UV light. If the compound of interest fluoresces this is a very sensitive detector. The analyte is excited by light commonly at 253.7 nm from a low pressure mercury lamp. The light is absorbed and the molecule is excited and it gives off light of a different wavelength. This wavelength is monitored by a detector which sits at right angles to the UV light source used to excite the analyte.

9. Electrical Conductivity The electrical conductivity detector was one of the first in-line detectors developed and is still in use for detecting electrically charged ions. It is based on the principle that the electrical conductivity of a liquid is proportional to the ionic concentration in the mobile phase. The design is simple, and the detector can be made small enough for an effective sensing volume of a few nanoliters, making this detector very useful for capillary electrophesis.

10. Refractive Index Detectors In contrast to the fluorescence detector, the refractive index detector is one of the least sensitive (about 1000 times less sensitive than UV detectors). Refractive index detectors are often used for the analysis of carbohydrates. Carbohydrates are generally not ionic do not fluoresce, and do not absorb UV or visible light to any great extent, so the refractive index is one of the few detectors that can detect them.

11. Light Scattering Detectors The flow from the column is converted into a fine spray or mist. The mobile phase is evaporated, leaving tiny particles of the analytes The particles are passed through a laser beam and they scatter the laser light. The scattered light is measured at right angles to the laser beam by a photodiode detector.

12. Mass Spectrometric Detectors For all mass spectrometer techniques, the analytes are first ionized since the detector only measures charged particles. The charged particles then enter the separator that separates ions on the basis of mass using a magnetic field. The masses of different fragments hit the detector where they give up their charge creating an electric signal which is recorded. It is difficult to use a mass spectrometer with liquid chromatography because the mobile phase must be removed before the analytes can enter the detector. A number of interfaces have been developed to solve this problem (ie., electrospray, ion spray, and thermospray) and LC-MS is becoming more common.