How to Perform an HPLC
Performing a HPLC (high performance liquid chromatography) test is a technical skill which should only be carried out by trained personnel. HPLC has been increasingly used in laboratories since the 1980s. It is used for research and in laboratories and universities to separate and identify complex organic molecules or compounds. The system can be run in normal or reversed phase.
The HPLC machine consists of a reservoir, degasser, injector, column attached to oven and detector, integrator and software (see Resource 1).
Other People Are Reading
Things You'll Need
- Software
- Computer
- HPLC
- Waste bucket
- Reagent (Methanol/Water)
- pH (litmus paper)
- Electricity
Instructions
-
Running HPLC
-
1
Turn on the HPLC machine following manufacturer's guidelines. Set up the instrumentation (see Reference 1). Setting the HPLC correctly is the principal aspect to this test and there is a standard technique. Choose the correct method for the substances to be analysed on HPLC.
Place the right column in the correct manner following the arrow showing placement. Set the wavelength to be able to detect the complex compounds. Set up the proper temperature.
Wash the column with 50% methanol and water for 30 minutes. Allow the mobile phase to pass through.
Set up the flow rate as stated in methodology.
-
2
The mobile phase of HPLC is critical and satisfactory results depend on whether preparation has been carried out correctly (see Reference 2). There are two types of mobile phase used in HPLC , gradient elution or isocratic. Gradient elution is where two organic mobile phases are needed, at different times. With isocratic elution, only one organic phase is required (methanol /water).
-
-
3
Ensure that the HPLC machine and computer are linked via an RS232 cable to allow information to flow between the two machines (see Reference 3).
-
4
Prepare the sample according to the specification in the methodology. Choose the specific analytical method desired and registered on the computer.
Choose the right HPLC when there are many linked HPLC machines. Go to instrument and open the sample list.
Open a window and write the name and date of the test. Save it.
-
5
The sample list will appear in chart form. Complete the details on the chart for the sample you have prepared.
Place sample vials correctly in the machine according to the methodology. Press start to begin the analysis.
-
1
Tips & Warnings
All steps in HPLC and the sample preparation must be correct in order to avoid a repeat test. Repeat investigations cost time and money.
References
Resources
- Photo Credit test tubes image by Ruta Saulyte from Fotolia.com
