Spectroscopy
Q. Write a short note on FRET (fluorescence resonance energy transfer).
Answer:
Fluorescence Resonance Energy Transfer (FRET) is a powerful technique used in microbiology to study the interactions between biomolecules, such as proteins and nucleic acids. FRET relies on the transfer of energy between two fluorescent molecules, a donor and an acceptor, when they are in close proximity. In FRET, the donor molecule is excited by a light source, which causes it to emit a fluorescent signal. If the acceptor molecule is within a certain distance of the donor, it can absorb some of the energy emitted by the donor and emit its own fluorescent signal. By measuring the intensity and wavelength of the acceptor signal, it is possible to determine the distance and orientation between the two molecules.
FRET has many applications in microbiology, including the study of protein-protein interactions, protein-nucleic acid interactions, and protein conformational changes. It is a valuable tool for understanding the molecular mechanisms underlying cellular processes and diseases, and it can be used in live cells to monitor changes in real-time
Q. Explain instrument of HPLC with suitable diagram.
Answer:
The Pump
-The pump is positioned in the most upper stream of the liquid chromatography system and generates a flow of eluent from the solvent reservoir into the system.
-High-pressure generation is a “standard” requirement of pumps besides which, it should also to be able to provide a consistent pressure at any condition and a controllable and reproducible flow rate.
-Most pumps used in current LC systems generate the flow by back-and-forth motion of a motor-driven piston (reciprocating pumps). Because of this piston motion, it produces “pulses”.
Injector
An injector is placed next to the pump.
The simplest method is to use a syringe, and the sample is introduced to the flow of eluent.
Column
The separation is performed inside the column.
The recent columns are often prepared in a stainless steel housing, instead of glass columns.
Detector
Separation of analytes is performed inside the column, whereas a detector is used to observe the obtained separation.
The composition of the eluent is consistent when no analyte is present. While the presence of analyte changes the composition of the eluent. What detector does is to measure these differences.
This difference is monitored as a form of an electronic signal. There are different types of detectors available.
Recorder
The change in eluent detected by a detector is in the form of an electronic signal, and thus it is still not visible to our eyes.
In older days, the pen (paper)-chart recorder was popularly used. Nowadays, a computer-based data processor (integrator) is more common.
There are various types of data processors; from a simple system consisting of the in-built printer and word processor while those with software that are specifically designed for an LC system which not only data acquisition but features like peak-fitting, baseline correction, automatic concentration calculation, molecular weight determination, etc.
Degasser
When gas is present in the eluent, this is detected as noise and causes an unstable baseline.
Degasser uses special polymer membrane tubing to remove gases.
The numerous very small pores on the surface of the polymer tube allow the air to go through while preventing any liquid to go through the pore.
Column Heater
The LC separation is often largely influenced by the column temperature.
In order to obtain repeatable results, it is important to keep consistent temperature conditions.Thus columns are generally kept inside the column oven (column heater).
Also for some analysis, such as sugar and organic acid, better resolutions can be obtained at elevated temperatures (50 to 80°C).
Thus columns are generally kept inside the column oven (column heater).
Q. Explain 2 detectors in HPLC & state their significance.
Answer: There are several types of detectors that can be used in HPLC to detect the separated compounds. Two commonly used detectors in HPLC are:
UV-Visible (UV-Vis) Detector:
The UV-Vis detector is a widely used detector in HPLC. It operates on the principle of the absorption of light by the compounds in the sample. The UV-Vis detector works by passing a beam of UV or visible light through the column effluent, and measuring the amount of light absorbed by the compounds in the sample. This detector is useful for detecting compounds that absorb UV or visible light, such as aromatic compounds and conjugated systems.
Mass Spectrometer (MS) Detector:
The Mass Spectrometer (MS) detector is a more advanced and sophisticated detector in HPLC. It operates on the principle of separating the ions of the compounds based on their mass-to-charge ratio (m/z). The MS detector works by ionizing the compounds in the sample and then separating them based on their m/z values. The separated ions are then detected by a detector that measures the m/z values of the ions. The MS detector is useful for detecting and identifying the compounds in the sample based on their mass spectra.
Significance:
The choice of detector in HPLC depends on the type of compounds being analyzed, the sensitivity required, and the complexity of the sample matrix. The UV-Vis detector is suitable for routine analyses of compounds that absorb UV or visible light, and it is widely used in pharmaceutical, environmental, and food industries. The MS detector, on the other hand, is useful for analyzing complex samples, such as natural products, metabolites, and impurities, and it is often used in research and development applications.
Monochromator: The monochromator is used to select a specific wavelength of light to pass through the sample. It consists of a prism or grating that separates the different wavelengths of light and selects the desired wavelength.
Sample holder: The sample holder holds the sample that is being analyzed. It is usually a cuvette made of quartz or glass.
Detector: The detector measures the intensity of the light that passes through the sample. The most common detectors are photomultiplier tubes (PMTs) or charge-coupled devices (CCDs).
Signal processor: The signal processor converts the electrical signal from the detector into a digital signal that can be displayed on a computer or chart recorder.
Display: The display shows the absorbance or transmittance of the sample at the selected wavelength.
Controls: The controls are used to adjust the wavelength, zero the instrument, and calibrate the instrument.
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