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.

UV / VIS, VWD, DAD, PDA HPLC DETECTOR SIGNAL BANDWIDTH (bw) SELECTION

Modern chromatography UV/VIS detectors offer the operator a choice of one to several hundred different signal wavelength choices (as is the case for Diode Array Detectors). Besides being able to specify a single wavelength, you can often choose a signal BANDWIDTH (bw) to associate with each wavelength [e.g. for a 280 nm signal with 10 nm bandwidth. This is often written as: 280 (10) or [280:10]. In many detectors, Signal Bandwidth is a variable, not fixed and represents the total number of nanometers across the specified signal value chosen. For example: If you select a signal wavelength of 280 nm and choose a bandwidth value of 10 nm, then you are actually gathering all signal data between 275 nm and 285 nm (5 nm to the left of the apex and 5 nm to the right for a total of 10 nm). Using a narrow bandwidth has the advantage of increasing the signal selectivity of the detector as you are only collecting data within a tight window. If you were to increase the bandwidth to 60 nm in the same example you would now be collecting data between 250 nm and 310 nm. The additional data collected over this wider range may reduce the total noise (by averaging it over a wide range), improve the S/N ratio (which may increase sensitivity), but it also reduces the selectivity. Large bandwidths also increase the chance you may include peak signal data from other co-eluting components into your signal data. You must select a bandwidth range for each signal wavelength which is located 'safely' away from any other potentially interfering peak. As with many things in life, balance is important. In this case, bandwidth choice is the balance between selectivity and sensitivity.

• When developing new methods we recommend that you choose an initial bandwidth value of 10 nm for each signal. This provides a nice balance between selectivity and sensitivity. It is also a common bandwidth value used on many older UV/VIS detectors which have a fixed signal bandwidth (such as many single or variable wavelength detectors).

• If you have determined the exact signal maximum for your sample and you would like to gain additional sensitivity for your sample (and thus decrease selectivity), re-run the analysis using several different, but increasing signal bandwidth values (e.g. 10, 20, 30, 50 and 100 nm). Choose bw values that are safely within the range of the detector, within the limits of the mobile phase's absorption region and also away from any potential co-eluting peaks. *To confirm which value is best, be sure and calculate the actual measured signal to noise ratio of the peak of interest after each analysis. This is a critical step! Do not be fooled by increases in the peak height or area alone as these changes are not always synonymous with better signal to noise ratios. Only by measuring the actual baseline noise level for each run and comparing it with the actual peak signal obtained will you be able to determine if increasing the bandwidth has provided you with better noise reduction and signal strength.

• To increase spectral signal selectivity choose a bw value that is very narrow. A value such as 2 or 4 nm would allow the detector to collect only signal data that is at or near the apex of your selected wavelength. This can be very useful when trying to discriminate your signal from nearby signal peaks, especially at low wavelengths such as 210 nm.

• When reporting your method conditions always include the wavelength AND bandwidth used for each signal. In order to accurately reproduce your method, this information is needed. *The flow cell dimensions, wavelength and bandwidth should always be included in your method.