1.2.2 High performance liquid chromatography (HPLC): HPLC is abbreviated as High Performance Liquid Chromatography or High Pressure Liquid Chromatography. In pharmaceutical and biomedical analysis HPLC has utmost feature that is for the development of the characteristic of the methodology since 25 years. During the process of discovery, development and manufacturing for the identification, qualification and quantification of drug analysis in active pharmaceutical Ingredient (API) or in the formulation, HPLC is the most important analytical tool. High Performance Liquid Chromatography (HPLC) is a one of the form of column chromatography that pumps solvent (called as mobile phase) and carried sample mixture or analyte into the column has chromatographic …show more content…
Generally, columns are packed with silica gel because its pores structure,surface properties and particle shape help to get a good separation. Various chemical compounds are separated by using silica due to its chromatographic behavior which is generally predictable and reproducible. Silica also has a higher surface activity which can be changed easily with water and other solvents. HPLC columns are classified into two, one is analytical column and the other is guard column. Typically the length of analytical columns are 5, 10, 15 and 25 cm and are filled with 3, 5 or 10 μm particle diameter. Usually the columns internal diameter is 4.6 mm. The guard column protects the analytical column and it is an essence a disposable top of the main analytical column. It increases the life of analytical column and protected from contaminants and particulate matter in solvents. The most popular material is octadecyl-silica means C18 column used in reverse phase HPLC and also C8, C6, C8, C4, cyano and amino columns are …show more content…
HPLC UV detectors used to detect and identify components showing an absorption spectrum in the UV or visible region (from 190–600nm). In UV detector, deuterium discharge lamp is used as a light source with the wavelength from 190-380 nm. Tungsten lamp is used additionally when components are detected at the wavelength exceeds 380 nm. When the light emitted from the lamp is focused on the grating, it scattered according to its wavelength. The diffraction grating angle is adjusted according to the required wavelength. Then that light passes through half mirror and it splits into two rays, one passes through reference-side light-receiving section and the other ray passes through flow cell. The difference in intensity of light can be determined between light from reference cell and flow cell, the output obtained as absorbance. UV detector detects all the components with high sensitivity. The schematic diagram of UV detector is shown in Fig. Fig.: The schematic diagram of UV
Before it can be used I had to calibrate the colorimeter. To do this I had to calibrate a colorimeter a cuvette filled with distilled water is placed into the colorimeter, the colorimeter in theory should then give a reading of the absorbance level being 0, this is because the water used in the calibration process is distilled and therefore should give a reading of 0, if not then it shows the equipment being used the experiment is either faulty or inaccurate. As all the light will pass
The HPLC method used was 20 μL of sample at 25°C through an RI detector for 15 minutes. The mobile phase was .0001M Sulfuric acid at .6ml/min with a column temperature of 60°C.
To understand this week’s experiment one must first understand what a spectroscope is and what it does. With this understanding in hand, one would gain a deeper appreciation for this lab and its intended lesson. “A spectroscope is a device that measures the spectrum of light” (Ball, 2014). More specifically a spectroscope is an instrument designed to split light from different sources into wavelengths. Humans are able to see these wavelengths as different colors. Noting the difference in colors between various light sources, those studying a given light source can identify elements of the light source.
Prepare silica gel column. Add 6 g of silica gel in 20 mL of hexane to make a slurry. Block column with small piece of glass wool, add 5 mL of hexane and then add the silica slurry up to the 10 cm mark.
At least 30 strips of paper- coffee filters or chromatography paper 3 cm by 9 cm
When the invisible UV light hits the coating of the bulb, the phosphor coating transforms the invisible light into light you can see. (U.S. EPA and U.S. DOE) There is no heating up of the filament, so the energy is usually not lost due to heat like when you use an incandescent lamp. The advantages and disadvantages of CFL will be researched in more detail in the next two tasks.
HPLC (High Performance Liquid Chromatography) is an analytical technique which separates a complex mixture of components into its specific individual components. It is a powerful tool in analysis, as it combines high speed with extreme sensitivity compared to traditional methods of chromatography because of the use of a pump which creates a high pressure and forces the mobile phase to move with the analyte in high speed. It is been used as a principle technology in various automated analyzers used for diagnostic purpose.
Liquid chromatography (LC) or more specifically known as high-performance liquid chromatography (HPLC) is a technique that makes use of chromatography to separate a mixture of complex compounds into its constituent molecules and can further be used to identify, quantify and purify these components.
A halogen bulb light source was set up at the end of a yardstick, and a light sensor was set up on the yardstick beginning at 0.1m from the light source. Science Workshop was again used to measure the intensity of light ranging from 0.1m to 1.00m (Fig.
Aim: The aim of this experiment is to measure the effect of different distances in centimeters, on the intensity of light using a voltmeter on a clear light bulb using a photovoltaic cells to detect the intensity of the light at different distances. The voltmeter will provide the readings of the light intensity with the Voltage as the unit of light intensity.
Outline the spots with a pencil. By putting the plate in an iodine chamber the spots can be visualized. Compound spots turn brown after a few minutes and then mark the spots after development in iodine vapor because the iodine color fades with time. The value of Rf can be calculated and unknowns components can be identified as shown in figure
The chloroform and methanol for sample preparation and the acetic acid (CH3COOH) used in the mobile phase were of A standard of analytical reagent (AR
A dichroic mirror is a special type of interference filter that efficiently reflects shorter wavelength (excitation) light and efficiently passes longer wavelength (fluorescent) light. It is oriented at 45O angle to the incoming excitation light path and reflects the excitation light at a 90O angle directly through the objective and onto the specimen (Fig.1). It also reflects any scattered excitation light back in the direction of the illuminator.
Thin layer chromatography is a classical case of adsorption or solid/liquid chromatography or planar chromatography. In planar chromatography, the stationary phase is applied on a flat surface and movement of mobile phase is due to the capillary action. The stationary phase is normally a polar absorbent and the mobile phase can be a single solvent or combination of solvents. Adsorption is a concentration dependent process and adsorption coefficient is not constant, in contrast to partition coefficient (liquid/liquid chromatography). Hence, if the concentration of sample is more than the absorptive capacity of stationary phase, the separation of components of the mixture will be poor. TLC is a useful tool for separating and identifying
HPLC gradient mixers must provide a very precise control of solvent composition to maintain a reproducible gradient profile. This can be complicated in HPLC by the small elution volumes required by many systems. It is much more