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In HPLC, how often should I need to run standard with samples? Why? If the retention time is changed with real samples compared to standard, how can I determine if the peak with changed retention time is samples are actually compounds A and B?

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In HPLC analysis, standards are run and their retention times are noted down in order to calibrate the instrument. Essentially this means running the calibration standards at the beginning of the sequence and at the end, and makes the assumption that any changes occurred in a linear manner during the sequence. The data system then changes the calibration incrementally from the beginning to the end, and applies this to the results.

Drift of peaks due to change in retention time is a major issue in HPLC. To identify the causes (shown in blue below) and remedies of these problems needs step-wise logical analysis:

(1) A new column may require several priming injections for the constituents of a sample to irreversibly bind to the most active surface silanol species - each injection being exposed to successively fewer silanol groups and hence producing a slightly different (typically shorter) retention time. To reduce the requirement for priming injections one should choose a high quality stationary phase, which uses Type B (type II) silica and which has a low metal ion content. To speed up the column 'equilibration' process, one should consider injecting a sample at 10x the usual concentration in order to quickly cover the surface silanol ...

Solution Summary

This solution offers details discussion and step-wise logical analysis of some major concerns related to HPLC, along with citation of some resourceful references.

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Questions about HPLC Lab

Hey I am about to do a lab to determine the amount of caffeine and sodium benzoate in soft drinks via HPLC. To prepare I had to answer a series of questions before the lab. I am confused on some of them and request your help. The questions are:

Why is it necessary to perform the HPLC separation at a pH of 3? How does the pH affect the separation of caffeine and sodium benzoate?

Predict the elution order for caffeine and sodium benzoate in a reversed phase HPLC used in the experiment?

Why is reverse phase HPLC is being used in this experiment?

At pH=4 what will be the charge if any of caffeine and benzoate?

Can you use an ion exchange HPLC column. If so, what ion exchange would be best?

What is the detection limit for a UV-vis detector?

Why aren't you concerned that the soda is colored or not, unlike the situation for the diode array experiment?

What would happen if I double the length of the HPLC?

When looking at the calibration curve, would I use peak heights pr peak area for accurate results?

Any help would be greatly appreciated.

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