Tips Bagi pemakai HPLC spesial merk Waters Wassalam Muh. Irwan HPLC TROUBLESHOOTING
There are some information for your troubleshooting in conection with the practical HPLC instrument. Althougt there is no substitute for experience or consulting with an"expert" here are some tips and tricks, some answer to the most commonly asked technical inquiris, and some key questions to ask to help diagnose our problem. But the most important we are following the WI to minimize the troubleshooting in our HPLC . 1. poor Peak shape, loss of resolution or peak splitting. � Probable cause. Mechanical fouling. Coloumn void. Sample overloud. Sample mobile phase incompatibility.dead volume from improperly assembled fittings and conectors. � Key question Are all peaks equally affected? Did the peak shape start out fine but degrade over time? Has the back pressure increased? � Becertain that; The mobile phase is the proper ph for your analytes. Your analytes are soluble in the mobile phase. You are not overloading the coloumn with to much sample. Ensure that the sample solvent is not to strong for the mobile phase. All connecting fittings are well seated. � First pass solution Make sure your coloumn is properly connected with appropriate fittings for perfect, zero dead volume conections. If the coloumn looks fine, then the problem is specifik to yur analyte or system. 2. high pressure � probable casuse; restricted flow-path � be certain that all components in the mobile phase and spl are compatible with one another, filtered, and that precipitation cannot occur. � First pass solution Replace column with a union and measured total system pressure. If presurre without coloumn is high, disconnect all conenections starting at the end of the flow path until you isolated the section giving high pressure. If pressure without coloumn is acceptable, attempt to remove particulate matter from the inlet frit by running the coloumn in reverse with the outlet disconnected from detector. If pressure is still high, refer to cleaning/regenaring coloumns procedure. Use inline filters or improve you spl preparation procedure to remove particulates and contaminating components. 3. Retention time or selectivity variation � Probable cause Variable pump flow, temperatur variation, or solvent mixing. Mobile phase preparation technique. � Be certain that Comparison are being made under identical system, mobile phase, and spl condition. The coloumn are properly equilibrate. Coloumn have not been exposed to conditions that could change them. Eqilibrium of coloumn using reagents has to be wel controlled. � Key question Is the void time consistent? Using the same spl and mobile phase bottle in the same day? � First pass solution Test the coloumn under our QA conditions to make sure they are stil in good conditions. 4. poor sensitivity � probable cause detector lamp energy low. Wrong wavelength. detector cell contaminated � be certain that HPLC system is clean, calibrated and operating properly. All liquid conections are zero dead volume. � key questions ; does asimiliar coloumn give better sensitivity? � First pass solution If you can get good sensitivity from another similar coloumn, adsorpsion may be occurring. The coloumn must to be cleaning and regenerations coloumn. 5. ghost peaks � probable causes contamination of coloumn or system � be certain that; the gost peaks is not from electronic noise � key questions what spl have you previously injected to the coloumn? Does the peaks diminish with each injection, stay the same, or get larger? � First pass solution; make several consecutive 0 uL injections. � If peaks decrease with each injection, the contamination is from aprevios sample injections. � If peaks stay the same size with each injection, the contamination is from mobile phase. � If no peaks is seen with 0 uL injections, the gosth peaks is your spl.
