External (ESTD) vs. Internal Standard (ISTD) Calibration in HPLC

Reliable quantitation of sample analytes using HPLC analysis requires accurate and reliable quantitation of a standard(s). For chromatography applications, we commonly use either an External Standard or an Internal Standard, as applicable, to insure reliable quantification of the sample.

• NOTE: A quick comment about calibration methods. Before you begin to create any calibration tables or analyze any standards/samples, please make sure that your current chromatography method follows good chromatography fundamentals. It must be selective for the sample type, retain the compound(s) with good K prime values, be reproducible and resolve apart all of the samples and possible impurities with near to perfectly symmetrical peak shapes. Your calibration results will only be as good as your original method. A poor quality method may not provide reliable results so be sure and spend as much time as possible developing the initial HPLC method to be as rugged and reliable as possible before starting any quantitation or calibration. *Poor quality method development is the number one reason for problems with quantitation.

Methods of Quantitation, Peak-height vs. Peak-area: Both types of response provide a measurement of the detector signal output. Proper and reproducible integration of the signal output is critical. Peak area is the most popular choice in chromatography, but peak height measurements can also be used if the peaks have near perfect symmetry (very rare, so peak area is far more reliable for integration). Whichever method you chose, you must use it consistently and document it well.

Definitions, External & Internal Standards: For most samples, there are two commonly used types of standards used. When known standards are run separately from the actual samples (in their own chromatogram) and their response is compared to that of the sample in another chromatogram, then we refer to this as an External Standard (ESTD). When the standard is added to the sample and analyzed at the same time we refer to this as an Internal Standard (ISTD). With an Internal Standard we are comparing the instrument's response to the sample to a reference standard with similar response characteristics, both run together.

External Standard (ESTD) Calibration Notes: The sample must fall within a range bracketed by the calibration solution. I suggest that you include a range which covers concentration values which are ~ 50% or more outside of the expected range. Dissolve the final calibration standards into the mobile phase (or a weaker solution) when preparing the injection vials from the stock solution. At least five (5) different concentration values should be used per order-of-magnitude (larger range = more stds). *Inject the same volume of solution (different concentration) for each calibration standard point ("level") onto the column. Do Not inject different volumes of solution from one std vial to create different concentrations. Plot peak response vs concentration. Ideally, you should have a linear response and the line will go through the origin (true zero intercept, ideally, though matrix effects/or the use on non std detectors such as the ELSD or CAD may require complex curve fits/formulas to describe the response). Once you have injected all of the standards, repeat the process again at least three more times (or use multiple injections) to determine overall reproducibility before constructing the final calibration table.

Internal Standard (ISTD) Calibration Notes: Internal standards are commonly used when many sample preparation steps are required before the sample can be injected onto the column. The internal standard may compensate for any losses during filtration or extraction. Selection of the Internal Standard is critical. Some of the characteristics of a good ISTD should include: It must be different than the sample, well resolved and must not elute where any sample peaks could be expected; It should not elute where any interfering matrix or other compounds could appear; It should have a similar linear response as the sample (Inject a fixed volume/concentration); Available in a high purity form from one or more commercial sources (certified method); Must be stable and not react with the sample or mobile phase solution. 

Add it to the samples before any extraction procedures. Base the amount of ISTD concentration such that it is between 1/3 and 1/2 of the expected concentration of the sample(s). The sample's target concentration range is a good value to use. *Because of these and other strict conditions, finding a suitable Internal Standard can take some time and testing.