PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Liquid Chromatography1. Introduction and Column Packing Material2. Retention Mechanisms in Liquid Chromatography4. Column Preparation6. Detectors(Chapter 4 and 5 in The essence of chromatography)3. Method Development5. General Instrumental aspectsDetectors in Liquid ChromatographyA. Refractive Index DetectorB. Absorption Detector (UV/Vis)C. Fluorescence DetectorD. Conductivity DetectorE. Electrochemical DetectorThese detectors differ from those used in GC in that most of them are nondestructive. This makes LC more attractive for purification or preparative-scale work that GC. LC does lack behind HC, however, in the fact that it does not currently have a good universals detector available for its use.A. Refractive Index Detector1. The RI detector is one of the few universal detector available in LC 2. Principle: The RI detectors measure a bulk property of the mobile phase leaving the column: its ability to refract to bend light (i.e., its refractive index). This property changes as the composition of the mobile phase changes, such as when solutes from the column. By detecting this change, the presence of solutes can be detected.3. Detector Design:i. One of simplest of RI detectors is the deflection RI detectorii. In this detector, light is created by a source and passed through flow-cells containing mobile phase eluting from the column (sample stream) and a reference stream (usually mobile phase with no solute in it). The light passing through these flow0cells is passed through a second time using a mirror and passed to a detector where its intensity is measured. iii. When the refractive index of liquid in the sample and reference flow-cell are the same, little or no bending of light occurs at the interface between the low-cells. This allows the largest amount of light possible to reach the detector.iv. As solute elute from the column, the refractive index of the liquid in the sample flow-cell will be different that that in the reference flow-cell and light will be bent as it passes between them. This changes the amount of light reaching the detector, producing a response.4. Applications:RI detector are universal applicable to the detection of any solute in LC. This makes them useful in preliminary work in LC where the nature or properties of a compound may not be known yet. They also the detector of choice for work with carbonhydrates or in the separation of polymer by size-exclusion chromatography. Some disadvantages: (1) they do not have very good limits of detection, (2) they can not used with gradient elution, where the composition of the mobile phase is changing with time. (3) The temperature of the system must also be controlled to avoid baseline fluctuations with these detectors. 5. SensitivityThe response of a RI detector is approximately the same for all compounds. 6. Limit of Detection: 10-5 to 10-6 M7. Linearity/ Dynamic Range: The response of a RI is usually linear over a 104-fold range in concentration.B. Absorbance Detector (UV/Vis)1. The absorbance detector is the most common type of detector in LC.2. Principle:Absorbance detector measures the ability of solutes to absorb light at a particular wavelength range. This absorbance is described by the Beer-Lambert Law. A = l c Where: A = Absorbance of light at a given wavelength Molar absorption coefficient of the solute l = path length of the flow-cell c = concentration of solute3. Detector design:i. There are three types of UV-Vis absorbance detector: fixed wavelength detectors, variable and diode array detector. They are generally based on the following type of design:ii. In a fixed wavelength detector, absorbance of only one given wavelength is monitored by the system at all time. The wavelength is usually 254 nm. A fixed wavelength detector is the simplest and cheapest of types of detector, but is limited in terms of it flexibility and the types of compounds it can used to monitor.iii. In a variable wavelength detector, a single wavelength is monitoredAt any given time, but any wavelength in a wide spectral range can be selected. The wavelengths that can be monitored can vary from 190 nm to 900 nm. The ability to use one instrument for more than one wavelength is achieved by adding in more advanced optics to the system.iv. Photo diode array detectors operate by simultaneously monitoring absorbance of solutes at different wavelength. The result is that an entire spectrum of a solute can be taken in a minimum amount of time.Advantages of Diode Array Detectorsv. Design of the cell5. Sensitivity: The response of an absorbance detector depends on the molar absorption coefficient. The larger this value is, the larger the response of the detector6. Limit of detector: 10-8 M 7. Linearity/Dynamic range: 105-fold range4. Applications:Absorbance detector can be used to detect any compound absorbing at the wavelength monitored. Absorbance detector can be sued with gradient elution.F = I (1-e- l c) = I l c (at low concentration) F = Fluorescence intensityI = intensity of the excitation light= Fluorescence quantum yieldMolar absorption coefficient of the solutel = path length of the flow-cellc = concentration of solute 2. Principle: C. Fluorescence Detector1. A fluorescence detector is an example of a selective detector, with limits of detection smaller than those by either RI or absorbance monitors.d. Applications: It can be used to detect any compound absorbing and emitting lightAt the given excitation and emission wavelength. c. Detector designe. Sensitivity: F = I (1-e- l c) = I l c (at low concentration)f. Limit of detection: 10-10 Mg. Linearity/Dynamic Range: 103 to 104-foldD. Conductivity Detector 1. A conductivity detector is an example of a ‘universal’ detector for ionic compound.2. Principle:i. This detector measures the ability of a solution to conduct a current when placed in an electrical field. This ability depends on the number of ions or ionic compounds present in the solution.ii. The relationship between the current, electric field and conductivity of the solution is shown as follows:I = C EI = CurrentC = conductivityE = electric field strength3. Detector Design4.