For internal standardization, a constant concentration of an internal standard is added to each sample and the ratio of the response of the analyte to that of the internal standard is compared with the concentration. In external standardization, the calibration is based upon comparing the response of the reference standard with its concentration. The two most popular methods of calibration are external and internal standardization. To do this, we use a calibration curve (also referred to as a standard curve or sometimes "line"). Nearly all of these methods rely on comparison of the peak area (or less often, peak height) of a sample with that of a reference standard. Most LC methods are used for quantitative analysis - that is, the methods that answer the "how much is there?" question. We also will look in more detail at normal errors as they relate to signal-to-noise (S/N) ratio in trace analysis. In future columns, we will look at how to determine the lower limits of a method as well as standardization techniques: internal standardization, external standardization and the method of standard additions. This month we will discuss some different calibration models, how to decide if a calibration curve goes through zero and some problems that can occur if the wrong choices are made. This month's instalment of "LC Troubleshooting" is the first of a series about calibration curves. The choice of the LOD and quantification is an important decision that can be confusing. Some of these problems relate to improperly selecting the model used for calibration. There seem to be a disproportionate number of problems encountered in the concentration region near the limit of detection (LOD) and lower limit of quantification with calibration curves used for liquid chromatography (LC) methods.
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