4.2: Analysis of Plasma Extracts and Drug samples by HPLC
- Page ID
- 364571
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B2: Analysis of Plasma Extracts and Drug samples by HPLC
Safety Notes:
The compounds used in these experiments as "unknown" samples are drug compounds and care must be taken to avoid ingestion and skin contact. The plasma solutions must be treated as biological hazards and all work must be carried out using gloves to avoid any contact. All biological wastes and contaminated glassware must be sterilized chemically after use (see technician).
Note the detector contains a UV source which must not be viewed directly
Apparatus
Reversed-phase HPLC system consisting of:
a Auto-sampler
b ODS (octadecyl silyl-) bonded silica column
c Pump set to deliver at 1 ml/min
d Mobile phase - methanol : water, 50:50
e Variable wavelength UV detector
f Integrator and printer
Method
- Check there is sufficient mobile phase in the sample reservoir and start the pump at 1 ml/min. Check the outlet tube is placed in the outlet bottle.
- The detector should already be turned on and set to 254 nm. Adjust the zero control so that an absorbance reading close to zero is displayed.
- Samples were prepared by diluting an aliquot of the plasma extract or drug sample with mobile phase and pipetting into vials. Load samples into auto-sampler taking note of the position and sample number.
- Follow the printed instructions next to the instrument (or consult a demonstrator) and start the analysis. Record the chromatograms.
- Compare the retention times of the peaks observed for the various samples and see if either of the plasma extracts correlate with any of the drug samples. At the end of the lab session, turn off the pump and detector, but PLEASE LEAVE THE INTEGRATOR SWITCHED ON.
Thinking About Making A Valid Measurement
You analyze a sample and get a peak at the same retention time as the standard.
How confident are you that the peak from the sample is the same as the standard?
Why are HPLC systems with mass spectrometer detectors becoming popular?
Improving Your Technique
- Choose a suitable column and mobile phase for the analysis.
- Use HPLC grade solvents.
- Mobile phase should be free of dust and dissolved gases removed - always use a solvent reservoir filter.
- Ensure there is sufficient mobile phase to complete the analysis.
- The sample to be analysed needs to be soluble in the mobile phase.
- Never use a syringe with a sharp tip because it will damage the injector.
- Investigate flow rate fluctuations because these may be due to blockages or leaks within the system.
- After use, always flush water, buffered solutions, acids etc. out of the system using an appropriate solvent.