Significance HPLC holds significance in the field of pharmaceuticals and medicine, in part, because these industries fall under

r the stringent regulations established by the U.S. Food and Drug Administration (FDA). If a company fails to test products using such methods as HPLC, FDA can issue 483 Warning Letters or even shut down a company's production via court order. High performance liquid chromatography is especially significant for the pharmaceutical and medicine industries because it allows for both qualitative and quantitative analysis. Benefits A benefit for a pharmaceutical company in using high performance liquid chromatography is the quality assurance this test provides. Another is that HPLC methods are relatively standard in the industry. This means new hires with experience in the pharmaceutical field require minimal training before they begin to perform testing in the laboratories. Additionally, the standard performance of HPLC testing enables contract manufacturers to transfer methods to their facilities from the company contracting production. Sponsored Links USA FDA Registrations Professional FDA Registration US Agent. No Separate Fee for Products www.FDAAgents.com Function HPLC is used to analyze raw materials and finished products to assure that pre-established quality levels are being met. HPLC begins as a high-pressure pump generates and meters a specified flow rate from a reservoir holding solvent. An injector introduces the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. A detector is used to measure the separated compound bands as they elute from the HPLC column. The mobile phase exits the detector and can be either collected or sent to wast What is an HPLC system? An HPLC system is unique among laboratory instruments because it can be assembled using components from different manufacturers and suppliers. Although many systems are sold as complete packages, a far greater number are assembled by bench level scientists and customized for specific needs. Pumps, detectors, columns, column ovens, and data management systems can all be interchanged. For example, when a pump is removed for service or the result of a breakdown, a new one can be swapped in with little or no interruption of service. Most components have their own keypad as well as a computer connection. The operator thus has a choice of running them off a central computer or via the keypads. Often the latter option proves the more convenient. Step 1, Preparing the System Column If the column mounted on the system is not the correct one for your assay, pump an appropriate storage liquid through the column before removing it. Cap the ends and return the column to the correct drawer. A column rack helps keep columns organized and makes them easy to find. Check the column's direction of flow. This is identified by an arrow stamped on the side of the column or marked on the label. After installing the correct column, tighten the connections so they do not leak. Firm but gentle pressure should be applied to the connections. Excessive force (such as you might need to apply while changing a flat tire) is not required. If you find yourself damaging the column nuts, switch to decaf and learn some relaxation exercises. Mobile Phase The mobile phase should be free of dissolved gasses so that no bubbles form inside the instrument during the run. Each system in the chromatography laboratory has a connection to a helium tank. Gently bubbling helium through the mobile phase for a few minutes will generally remove all dissolved gasses.

Some laboratories use sonic baths to degas the mobile phases and many new HPLC systems are sold with degassing units built into the pump. Often the mobile phase is mixed in large quantities. When preparing a mobile phase with two or more components, be aware that large amounts of heat can be generated by the mixing process. Glass bottles may crack! The selection of mobile phases is based on the relative polarity of the analytes and the column packing. Often the addition of just a few milliliters of some ionic species will dramatically affect the analysis. For example 20mM triethylamine in the mobile phase will reduce certain types of peak tailing. Adding acetic acid to a mobile phase will increase its overall polarity and result in better separations when running on a non polar stationary phase. Controlling the mobile phase's pH by the addition of acid, buffer, or base will often improve reproducibility. Step 2, Prime the pump. Select your mobile phase by either placing your intake line in the bottle or using the keypad on the pump to select a specific bottle. Open the prime or the purge valve on the pump module. Place a beaker under the outlet. Activate the prime or purge function on the pump. If the pump does not have this function, turn up the flow and switch it on. Run the system for a few minutes. When the pump is properly primed, the system will deliver a smooth flow of mobile phase from the outlet, produce a steady sound with no burping or grinding, and the inlet line will be free of air bubbles. Turn off the pump and close the valve. Step 3, Set the detector wavelength . Turn on the detector power and allow the unit to warm up. Using the keypad or the control computer GUI, set the wavelength. Step 4, Start the mobile phase flow. Use the pump controller, to set the flow rate for the mobile phase. Restart the pump. Watch the system's pressure indicator or gauges (if any) to see that it does not exceed the maximum allowed for the various components. If pressures become too high, slow down the flow rate. If pressure continues to rise, turn off the pump, and perform the following procedures: a. If the system has a guard column, replace it. b. The analytical column may need to be back flushed. Remove the column and reverse it so that mobile phase flows through it in the wrong direction. Catch the outflow in a beaker instead of allowing it to enter the detector. To condition the column, run mobile phase through it for a few minutes. Some operators recycle their mobile phase by running the waste line outlet back into their mobile phase reservoir. Care must be taken to avoid contaminating the reservoir with old samples, or impurities from the column. Monitor the detector output, when the signal is stable, you can begin running the samples.

Step 5, Inject the samples. Before injecting your sample, check the needle's tip. HPLC Needles have a smooth or blunt tip. Do not use a needle with a sharp tip or a tip with metal burrs. These will scratch the inner surfaces of the injector and cause it to leak. Remember this is a two-step process; syringe injection followed by turning the valve from "load" to "inject." Open the injection valves by turning them to load. Insert needle into the plastic needle guide as far as it will go. Smoothly inject the appropriate amount of sample. After the syringe is completely empty, quickly and smoothly turn the valve to inject. You may safely leave the syringe in place. Start the data system recording. Behind each injector, there is a small coil of tubing. Known as the "sample loop," it holds the sample during the interval between the syringe injection and the start of the run. The sample loop can be identified by a small tag listing its volume. When the injector is set to the 'load" position this loop is isolated from the mobile phase flow. Turning the valve to "inject" diverts the mobile phase flow through the sample loop and sweeps the sample onto the column. Sample loop size is very important when using an autosampler. For manual injections, as long as the sample volume is less than or equal to the loop volume, changing the loop is not necessary. As you become more experienced with HPLC, you will learn how to determine a good injection volume. Column capacity, detector sensitivity, and column size, must all be balanced to obtain the best results.

Hints for good injections: a. Make sure there are no air bubbles in the syringe. Even a small bubble can affect the injection volume. Also make sure that any excess sample is wiped off the exterior of the needle. b. Insert the needle completely into the injector's plastic sleeve. Do not press the plunger until the needle can go no further. c. Inject completely before turning the valve, but do not wait more than a few seconds between these two operations.

Step 6, After the run. Stop the data station. Return the injector to the "load" position and remove the syringe. Step 7, Cleanup. Turn off the detector, pump, and any other components. Rinse any sample residues from the syringe. Record your sample, date and initials in the appropriate notebook. Label your chromatogram with: 1. Your name, date and sample ID.

2. The column, mobile phase, flow rate, and detector wavelength. 3. The system number and its calibration date (if any.) Step 8, Is this data any good? Evaluate the chromatogram, look for: 1. Good separation. Is the first analyte of interest sufficiently separated from the solvent front? Are all analytes separated or are peaks running into each other? 2. Peak broadening and tailing. Broad peaks are difficult to quantify. Excessive tailing can hide minor peaks. 3. Irregular retention times. Retention times should not shift from one run to the next although small shifts caused by operator variability are normal. 4. Flat and level baselines. Undulating, irregular, or jagged baselines indicate contamination of the column, carry over between injections, or excessive sample loading. Similar symptoms can result from problems with the detector.