ESTD vs ISTD

Question

071619

Hi,

Which calculation method is better in Chemstation External standard calculation or Internal standard calculation? Is there a way to utilize both for better accuracy and precision? Any relevant response is welcome. Thanks!

schlereth1974 · Answer

Hi megaflorch,

First of, a bit of explanation.

External calibration:

You have got one ore more standards with known concentration. You are using these to connect the measured response (mainly peak area) to the amount of substance in your standard. The resulting calibration is used to calculate the amount of substance in an unknown sample.

Internal calibration:

You have got one ore more standards with known concentration. You are using these to connect the measured response (mainly peak area) to the amount of substance in your standard. The resulting calibration is used to calculate the amount of substance in an unknown sample.

ADDITIONALLY you have got a substance in all standards, blanks and samples, preferably at the same concentration.

The substance must not be part of your sample, but should be as similar as possible. In high end MS, deuterated substances are often used.
This substance is going to be added before sample preparation. If you are losing some of your substance due to sample preparation you have lost some of the internal standard as well and you can use this known loss to compensate the other unknown amounts in calculation.

So, Internal Calibration essentially already includes external calibration.

Internal Calibration usually give you better results if you sample undergoes extensive preparation steps with a lot of possibilities to lose substance.

Prerequisites are that you do have a substance you can use as internal standard.

I hope this helps.

Best regards,

Dominik

crystalnipp · Answer

Hello! I wouldn't say either is inherently better. Each has benefits and disadvantages. We have many methods (LC and GC) that are ESTD and many that are ISTD. I'd say that the main issue we have had with ESTD is that injection amounts need to be reliably accurate and consistent. So cheap sloppy syringes, poor injection technique (if manual injection), partially plugged syringes, poorly maintained injectors, less than optimum split flow control can pose problems. For ISTD's sample preparation is critical as you need to add an exact and known amount of ISTD. We have problems with this when people get sloppy with sample prep. It can also be a challenge to find an appropriate ISTD. It can't co-elute with anything in your sample, it can't react with anything in your sample, it has to be stable, it has to be compatible with your sample prep (soluble in the matrix). It has to be detectable via the mode you use. When I am setting a method up and I have the option of choosing the internal standard myself, I try to select one that is chemically similar the the compound(s) that I am trying to quantify. And elutes near the compound(s) of interest. You can definitely use both quantification methods, but that would require a custom report or some type of add on macro to have it generate automatically.

trifecta · Answer

Hi Megaflorch, I am commenting based solely on GC/MS experience. ISTD calibration has always given more accurate answers for the application areas we have dealt in. These include environmental, pesticides and drugs. In most cases these represent complex matrices, a large number of analytes and a wide calibration range. We use multiple ISTDs (labeled) at a fixed concentration. This mix is added to every standard, sample, blank etc. final vial contents, at 10 uL / 1mL . We do not track recoveries using the ISTDs as they are added at the end. Recoveries are tracked using surrogate ( or spike ) standards that are added at the start of the sample prep process. We can isolate sample prep issues separate from instrument performance issues with this approach. Accurate ISTD addition is easy using a 10 uL autosampler syringe. Agilent GC autoinjectors can do a sandwich injection of your vial and a separate ISTD vial, automatically, if you don't want to go manual. Data analysis is only slightly more complex as you need to add ISTDs and concentrations to your method. I don't see the value in doing ESTD if you do ISTD. Best Regards, Trifecta

ESTD vs ISTD

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