Why we have to calibrate the hplc?

Answer 1

1.calibration of pump.

2.column themostate /oven calibration

3.gradient composition

4.accuracy of autoinjector and detector response

5.carry over test.

6.wavelength accuracy

7.system precision.

Answer 2

This is an extremely simplified overview of the process assuming the GC has been programmed appropriately with proper oven temperatures, flow rates and timings.

Typically gas chromatographs are calibrated using a mixture of compounds of known concentrations. One example of this would be a "Standard Gas" which is specially mixed by a supplier to the customer's specifications to match component gasses that they would be testing for. As an example, 21% Oxygen, 75% Nitrogen, 2% Carbon Dioxide and 2% Carbon Monoxide might be used if the GC is to be used for testing breathing air. After being mixed, the supplier samples the gas and measures it on their own test equipment and issues a tracable certificate with the Standard Gas that allows the tracing of standards used all the way back to the metric standard weights and measures.

A chromatography run is then performed on the GC watching for peaks on all the various detectors. Once the run has been completed, the peaks for each compound are isolated, by the operator, and a cursor is placed to bracket each peak. The idea is to tell the GC where on the chromatography graph to start and stop calculating the area under each peak.

Once these cursors are placed, the GC calculates the area under each peak in the graph, and the operator informs the GC what the concentration of each component gas was in the Standard Gas, and the GC uses simple math from then on to calculate what a given component concentration in a sample is based on the pixel count under the peak for each component.

When running a sample, a base-line is first run using the Standard Gas, and the values compared to the Standard Gas's certificate values. If they agree, then you're ready to run a sample. The sample is run, and the results produced, but they are not final until a second base-line run is performed. If the values from the first and second base-line runs match, then the calibration on the instrument hasn't changed, and the results from the sample run are valid. Multiple samples can be batched between base-line runs, but their results should not be considered valid until a base-line has been run both some time before and some time after the sample(s).

Answer 3

This is a difficult question to answer because there are so many uses for the HPLC.

The HPLC is widely used to separate a complex mixture into individual compounds. Scientists use standards in order quantify the amount of a particular compound they have by generating calibration curves.

How all this is done...

Choose an eluent that will separate your compounds - test out different eluents on a TLC plate to make sure that your compounds are separating.

Set the temperature on the HPLC so that it is high enough to evaporate the eluent.

Insert your sample into the injection port (typically around 20 uL). Wait for your peaks to all come out. There are different ways to miminimize the amount it takes to run samples, but this is unique to your experiment, and so you have to just try out different things.