Hplc detectors

Hplc detectors

1. HPLC detectors Presented by: IRFAN AHMED PHARM.D 3 rd proff . Akhtar saeed college of pharmaceutical sciences,lahore

2. HPLC detectors INTRODCTION Detectors with high sensitivity are required in HPLC, usually with sensitivities in microgrames to nanogrames . There are following types of detectors which are commonly used 1)differential refractometer detector 2)ultraviolet detector 3)Diode array detector

3. Detectors 4)Fluorescence detector 5) amperometric detector

4. Differential refractometer detector It is often called as universal detector Principle This measures changes in refractive index of the eluent that result from the presence of solutes as they emerges from the column Draw back It can not be used effectively gradient elution due to a change in baseline(a change in the solvent index of refraction as the gradient is change) nor when the solvent has an index of refraction close to that of solute This detector is very sensitive to temperature This can detect concentrations of about 10-5 to10-6g\ml.

5. Differential refractometer detector

6. The ultraviolet detectors They have much better sensitivity ,about 10-8g\ml It is not temperature sensitive It is relatively inexpensive and can be used with gradient elution It is sensitive to a large number of organic compounds It can not be used with solvents that have significant absorption with ultraviolet or with sample components that do not absorb in u.v many UV detectors are simple interference devices that can measure only a few selected wavelengths

7. The ultraviolet detectors The more expensive detector have a monochromator that allows the selection of particular wavelength. Scanning of spectrum can even be achieved for qualitative identification by momenterily stopping the flow of the mobile phase The most popular HPLC detector is variable wavelength UV- vis detector It can measure nanogram amounts of UV absorbing analytes or those with suitable chromophores that absorb in the visible region

8. UV DETCTOR

9. Diode array detector It is a common feature of modern HPLC instruments The instantaneous recording of absorption spectra provide a powerful qualitative tool Principle The focused radiation source passess through the detector flow cell and is dispersed by a grating to a photo diode array for detection The ability to mathematically resolve overlapping spectra can provide additional separating ability when a chromatographic peak may consist of two or more analyte

10. Diode array detectors

11. Fluorescence detector It can give improved selectivity over UV absorption detector b\z fewer compounds fluoresce then absorb Sensitivities are at least good as perhaps better then UV detector achieved Depending upon the geometry of excitation of source-detector arrangement , the intensity of the source .

12. The amperometric detector It is useful for detecting electroactive substances and has found considerable use in biological applications Example In the HPLC separation and detection of trace quantities of catecholamines from brain In arranging the apparatus ,there must be minimum of dead volume b\w injection port and column and b\w column and detector to minimize spreading of peaks and to obtain maximum efficiency

13. The amperometric detector A 20cm length of stainless steel capillary tubing can generally be used to connect the column to the detectors without significantly affecting column performance The detector volume must be small and typical volumes are on the order of 1uL or so,with high performance detector used with capillary LC systems having submicroliter volumes

14. THANX