High Performance Liquid Chromatography - Lab Report Example

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY How A HPLC Unit Operates The use of isocratic high performance liquid chromatography (HPLC) entails the analyte being passed through the stationary phase. The liquid mobile phase provides the force that enables the analyte to pass through the stationary phase because it is pumped at a high pressure in the column. A small amount of the sample undergoing analysis is placed in the mobile phase where its movement up the stationary phase is influenced by the chemical and physical reactions that occur with the matter in the stationary phase. The movement of the analyte is dependent on its composition, the mobile phase and the stationary phase. The retention time is the amount of time it takes for the analyte to elute and is unique in each sample. The linear velocity that is enhanced by the high pressure that is used gives the components minimal time to diffuse inside the column. This is necessary because it allows for a higher resolution of the chromatogram that is used to deduce results. In a typical HPLC, various miscible organic liquids are used and the modern units have the capacity to generate a maximum pressure of 6000psi. HPLC units are available commercially and the buyer selects a type that is most suitable for their need. Different types come with different detectors, for example, UV absorption detector, refractive index detector, conductivity detector, evaporative light scattering detector, fluorescence detector, and electrochemical detector. Below is a representation of the HPLC unit that is described above; Source: Skoog, Holler, & Crouch, 2007. The Application of HPLC Units Generally, there are two types of HPLC units, one being the preparative HPLC and the other an analytical HPLC. In the preparative type, compounds are usually isolated and purified whereas the analytical mainly get detailed information about a sample that is passed through it. This means that the sample can be identified, quantified and its resolution obtained. The main uses of HPLC, however, are identification and purification of samples. HPLC is highly preferred by many professionals because it is accurate, sensitive, simple to use and can analyze nonvolatile samples that are sensitive to temperature changes. Furthermore, HPLC can easily be used in combination with mass spectrophotometry as means of validating the data collected. Most chemists use the technique in the analysis of amino acids, hydrocarbon, proteins, pesticides, pharmaceuticals, among other products. Safety Precautions When Using HPLC It is recommended that all persons operating the HPLC unit in the laboratory to wear protective nitrile gloves and a lab coat. Preparation of the Sample A combination of 0.7mg/ml phenol, 4.0mg/ml toluene and 5.0mg/ml ethyl benzene is dissolved in 10ml isopropanol. The amount of each of the above components is then calculated accordingly to allow for later calculations. The calculations for 4.0mg/ml Toulene, for example, are; 10 ml of 40mg Toulene are needed Its density s 0.87mg/ml The volume needed, therefore, is deduced form dividing mass over density i.e. 0.04g/(0.87g/ml) The result is 0.05ml (5 micro liters) Method The Dionex Ultimate 3000 HPLC that is made of a pump, columns and detector is used in this case. The steps that must be followed when using the unit are listed below; 1. First, start the computer that is attached to the HPLC unit. 2. On the “Chromeleon” icon that is on the desktop, double click so as to connect to the Chromeleon server that is under my computer icon to which okay is clicked. 3. Go to the view tab at the top of the screen and choose the default panel tab set. 4. On the main panel, select the home tab and connect all so that the pump, the column and the detector are all connected to the Chromeleon server. 5. On the main panel, click on the pump tab and then connect. The triangular indicator that is next to the connect option should be green. 6. Go to the motor option and switch it on. 7. Open the top panel on the instrument and loosen the yellow valve so as to purge it. The purging should be done for 5 minutes after which the yellow valve is tightened to complete the purging. 8. Under the main panel, select the DAD tab to switch on the lamps. 9. If the Alltech Altima silica is not in place, insert it in the column section of the apparatus. When doing so, conform that the green arrow in the column points towards the left side. 10. Once the pressure becomes stable following the completion of purging, use the pump tab to set the flow to 1.0ml per minute. At the most, the pressure should be 2000psi. Set up the waste container appropriately. Creating A New Program File To create a new program file, follow the steps below; 1. Click on File followed by New, Program File and Okay. 2. The screen after the above actions should show Temperature Control. 3. Click on Next whose screen should display the Column Oven Options. Choose Column A then select Pump and specify the amount of pressure needed. 4. Click next to bring the Pump Options and set the Gradient Type to Isocratic. Put the start of %B Solvents at 80.0%. 5. Click on Next to bring the Acquisition Options and set the Acquisition Time from 0 to 15 minutes. 6. Click next to display the Pump Pressure Options and set it to Auto. Select Average. 7. Click on Next to create a title for the program and Save. Creating New Sequence and Method File To create a new sequence and method file, follow these steps; 1. Go to File followed by New then Sequence using wizard then Okay. 2. The wizard should offer a guide to making a new sequence and the parameters do not require changing. Simply make a Title for the sequence and save. 3. To create a new method, click on File followed by New, Method File and Okay. 4. Make a title for the method and Save. The parameters do not need alterations. 5. If needed, do a pretest of the method. 6. Load the sequence and the method through selecting the files created. Sample Injection To inject the sample into the HPLC unit, the following steps are used; 1. Prior to injecting the sample, conform that all other conditions of the apparatus are well set. Click on the Batch option that is located at the top of the Default Panel Tabset window and click Start Batch. 2. After ascertaining that the HPLC is in proper condition for use, pull a 5 micro liter sample into a syringe with a capacity of 10 micro liters. Inject the sample with the lever being in the Load position then remove the syringe. 3. Click Start to pull the lever down so as to inject the sample after which it moves to the Load position. 4. It will take 15 minutes for the chromatogram to show as set in the time limit. Viewing and Recording the Data 1. Once the data collection is complete, click File followed by Batch Report. 2. Export the data using the preferred format. Switching off the HPLC Unit To turn off the HPLC apparatus, use the following steps; 1. Go to the Default Panel Tabset, click Pump and change the rate of flow to 0 micro liters per minute. 2. Click the DAD Tab and turn off the lamps. 3. On the Pump Tab, turn off the Motor. 4. Go to the Home Tab and select Disconnect All. 5. On the apparatus, switch off the main compartments manually. Works Cited Skoog, D. A., Holler, F. J., Crouch, S.R. Principles of Instrumental Analysis, 6th Ed. Thompson Brooks/Col., 2007. 816-826. Read More