We know that a substance can be classified as element, compound and mixture. Element and compounds are pure forms of any substance whereas mixture is formed by mixing of more than one component. On the basis of uniformity of mixtures, they can be classified as homogenous and heterogeneous mixtures. Homogenous mixtures have uniform distribution of all the components throughout the mixture.
They are also called as solutions. Heterogeneous mixtures do not have uniform distribution of components. Colloidal solutions and suspensions are examples of heterogeneous mixture. We have several separation methods which can be used for the separation of components of a mixture. These methods are based on different properties of components such as boiling point, solubility etc. Some common methods of separation of components of mixture are filtration, distillation, separating funnel, chromatography etc. Filtration is a simple separation method which is based on the solubility of components. Different types of filter papers for this method. Distillation is separation method based on difference in boiling point whereas chromatography is based on solubility. Chromatography is one of the best analytical separation methods which can be used to separate the mixture into its individual components. On the basis of different principals, they can be classified as liquid chromatography (LC), high pressure liquid chromatography (HPLC), gas chromatography (GS), ion-exchange chromatography (IEC), affinity chromatography etc. High performance liquid chromatography. Basically it is a type of column chromatography. In basic column chromatography, a solvent is dripped through a column under gravity whereas in HPLC, the solvent is forced at high pressures into the column.
The pressure in HPLC is close to 400 atm that enhance the speed of process and provided accurate results in short time period. Apart from high pressure another modification in HPLC is the use of smallest particle size for the column packing material. It increases the surface area for interactions between the stationary phase and the mobile molecules. It allows a much better separation of the components of the given mixture. Another improvement in HPLC is the use of highly automated and extremely sensitive detectors.
The detector in chromatography involves in identification and measurement of concentration of eluting components in the mobile phase. Today we have a broad range of detectors for different samples. When detector interacts with particular compound, it responds for the mobile phase composition. Some bulk property detectors also show respond on changes in composition of sample and mobile phase. Overall a detector should exhibit sensitivity towards solute over mobile phase, low cell volumes that can minimize memory effects, low noise and detection limits with large linear dynamic range. Different types of detectors are used for in HPLC which are based on different principals such as ultra-violet absorption etc. In UV absorption detector, the UV radiations are passed through the sample solution and the amount of light is detected by detector on other side. The absorbed light depends on concentration of sample solution. Here the used solvent can also absorb certain wavelength of light so we have to use the wavelength greater than the wavelength of solvent to avoid the false reading.
In HPLC, the separation of component in the sample is carried inside a column so it is an example of column chromatography. The separation process can be detected with the help of different detectors in which components are monitored and expressed electronically. Some common types of HPLC detectors are listed below.
|| HPLC detectors
|| Common Abbreviation
|| UV detector
|| Visible detector
|| PDA detector
|| Multi-Angle light scattering detector
|| Optical rotation detector
|| Electrochemical detector
|| Refractive-Index detector
|| Mass spectrometer
|| Conductivity detector
|| Fluorescence detector
|| Chemi-luminescence detector
|| Evaporative light scattering detector
Agilent HPLC detectors are new diode-array detectors which can work for multi-wavelength in HPLC. They are basically a type of photodiode array (PDA) detectors and widely used in analytical laboratories to increase productivity with good data quality. PDA detectors work with signal sampling rate of 20 Hz and a spectral sampling rate of 10 Hz which are not fast enough to keep up with the speed of separation. Agilent HPLC detectors work on multiple-wavelength and full spectral detection at a spectral sampling rate of 80 Hz. The Agilent 1100 Series have additional advantages of data security and traceability so it prevents data loss in case of communication breakdowns.
- UV- visible detector: They are the most commonly used detectors in HPLC. The amount of light absorbed by the given sample determines the response of these detectors. The intensity of light absorbed depends on the presence of functional groups in the molecule.
- Photo diode array detector: Such HPLC detectors have large number of diodes which act as detector elements. The presence of diodes makes possible simultaneous monitoring of more than one component at multiple wavelengths. It is one of the time saver and cost effective detector for HPLC.
- Fluorescence detector: Compare to UV-Vis, this detector is more sensitive but only effective for naturally occurring fluorescent compounds. They can use for other compounds with the help of post column derivatization.
- Mass spectroscopic detector: It is one of most sensitive and selective detector which is based on fragmentation of molecules in the presence of electric fields. The separation in this technique occurs on basis of m/z ratios of fragmented molecules.
- Refractive index detector: It is a type of bulk property detectors. The fluctuation in refractive index of eluting compounds determines the response of such detectors. Here the refractive index of mobile phase and sample should be different. These detectors are less sensitive than UV-VIS detector and also work on several limitations like temperature control.
- Electrochemical detector: It is also a type of bulk property detectors. Such detectors are based on electrochemical redox reactions of sample on electrode surface.
- Light scattering detectors: They are other types of bulk property detectors which are mainly used to detect high molecular weight molecules.
- Evaporative light scattering detector: They are sensitive detectors which are widely used for non-volatile analytes. It can be used for the gradient method and gives sensitive detection with stable base line.
- Conductivity detector: Such detectors work for ionic compounds as they are good conductor of electricity. It measures electronic resistance which is directly proportional to the concentration of ions in the solution.
- Chemiluminescence detector: In such detectors the excitation of molecules is initiated by chemical reaction. They provide higher sensitivity than Fluorescence detector.
- Optical rotation detector: These detectors are mainly used for optical isomer measurement. The separation of R- and L- type optical isomers can be done in column and later detected by these detectors which are not possible with other ones.