How do you troubleshoot an HPLC?
How do you troubleshoot an HPLC?
- Start pump.
- Check mobile phase level in reservoir(s). Check flow throughout system. Examine sample loop for obstruction or air lock.
- Check system for loose fittings. Check pump for leaks, salt buildup, unusual noises.
- Disconnect tubing at guard column (if present) or analytical column inlet. Check for flow.
What is the reason for area variation in HPLC?
The most likely source of variation is the composition of the mobile phase. In reversed phase chromatography, there is an exponential relationship between the retention factor k and the volume fraction of the organic solvent in the mobile phase.
What affects peak area in HPLC?
The peak capacity of a chromatographic system is shown to depend on the column efficiency and the capacity ratio of the most retarded solute.
How do you resolve overlapping peaks in HPLC?
Peaks that are moderately overlapped can often be resolved by increasing column efficiency — by increasing the column plate number to sharpen the peaks (reduce peak volumes).
What causes low pressure in HPLC?
Low pressure usually results from air in the pump, a faulty check valve, or a leak. First, check for the obvious: make sure the flow rate is set properly and that there is sufficient mobile phase in the reservoirs.
What causes negative peaks in HPLC?
Negative peaks are most often caused by difference in refractive index between the sample solvent, sample and mobile phase. They are also caused after routine maintenance when the system has not been reconfigured correctly.
What is void volume in HPLC?
Void volume refers specifically to the volume of the liquid phase contained inside a column. The same term is sometimes also used informally to refer to the volume of a cavity in the column/tubing or fittings. Void volume is also known as dead volume.
What is area ratio in HPLC?
The area of a peak is proportional to amount of the compound that is present. The area can be approximated by treating the peak as a triangle. The area of a triangle is calculated by multiplying the height of the peak times its width at half height. In addition, the ratio of B to A can be found using the areas.
Does Peak area have units?
However it is measured, the units of peak area are the product of the x and y units. Thus, in a chromatogram where the x is time in minutes and y is volts, the area is in volts-minute. In absorption spectrum where the x is nm (nanometers) and y is absorbance, the area has the units of absorbance-nm.
How do you improve tailing factor in HPLC?
There are a few methods that can be used to avoid peak tailing:
- Operate at a lower pH.
- Use a highly deactivated column.
- Consider the possibility of mass overload.
- Consider the possibility of column bed deformation.
- Work at high pH when analyzing basic compounds.
- Use a sample clean-up procedure.
What is Ghost Peak?
Ghost peaks are of unknown origin in a chromatogram, are easily misidentified when they are close to peaks of interest, and can result in quantitative errors when they overlap peaks of interest. Uncertainty in data quality and reliability is of course the result.
How do you calculate purity in HPLC?
Prepare a known concentration of your sample. Run HPLC, calculate peak area and concentration from the calibration curve. Purity = (concentration calculated/prepared concentration)* 100% First prepapre the calibration curve using standard solutions of the analyte in a particular mobile phase.
What causes peak tailing in RP HPLC?
1.1. Peak tailinghas been the most common peak shape problem since the early days of RP-HPLC. Most peak tailing is due to interaction with acidic or ionized silanol groups on the surface of the silica particles within the column. The low-purity silica (often called “Type-A” or acidic silica) has a high content of
How to calculate Peak area and concentration from the calibration curve?
Run HPLC, calculate peak area and concentration from the calibration curve. Purity = (concentration calculated/prepared concentration)* 100% First prepapre the calibration curve using standard solutions of the analyte in a particular mobile phase. Then take extracted or purified sample’s HPLC chromatogram for its peak at the retention time.
How can I determine the purity of my sample?
The only way to get an accurate purity is to compare your sample to a standard of well known concentration and composition analyzed in the same sequence of runs. However, many compounds do not have well characterized standards available so that is difficult.
