Internal Standard (ISTD) HPLC Calculation Notes:

Why Use an ISTD?

Two main reasons: (1) The addition of an internal standard to all vials containing standards and any unknown samples takes into account any changes caused by the sample preparation process. This is useful when the samples are run through various media or filters as part of a pre-treatment or clean-up initial phase (e.g. clinical or biological samples). (2) Retention time drift over the day can be compensated for through the use of ISTDs.

Key Points To Keep In Mind: 

• The ISTD must be of known purity (by certified method), have a similar response as the sample and not interfere with the analysis.

•  The calculated amount of your unknown samples is directly related to the amount of the ISTD used. The results are calculated based on the ratio of the responses for both peaks (i.e. std and unknown). The amount of ISTD used in all vials must be kept constant.

• Relating peak retention times based on the ISTD (as a ratio) instead of establishing retention time windows makes it much easier to transfer methods to other systems and also account for variation seen. This is because with ISTD and Relative Response Ratios you can define peak retention based on the elution time of the ISTD and not the actual retention times. 

• You must use identical integration parameters to calculate the areas of the standards, samples and/or unknowns. Failure to do this may invalidate the process.

• For Multi-level Calibrations you will calculate an amount and response ratio. You will do this for each calibration level (and each std type if multiple standards are used). Note: Most professional chromatography data systems are designed with special fields for the internal standard data and will perform these calculations for you once you load the data for each level into the calibration table. Response ratios are then used for measurements (Response Ratio = Sample/Std Area / ISTD Area). The accuracy of your calibration curve fit and the overall reproducibility of the entire method used will impact your final results. Poor quality curve fit and/or RSD equals poor accuracy.

• Highest accuracy is achieved using a professionally developed method which first retains (with proper K prime values), then elutes all samples off the column during the run. Column wash and equilibration steps should be separate runs, not part of the analysis method. Poor quality method development is the most common reason why calibration results are poor or RSD is high. 

• Use the same injection volume for all samples (unknowns) and standards (knowns).

• 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 range is best.

Calculations:

Response Ratio = Sample Response (Area) / ISTD Response (Area)

Response Factor = Amount Ratio / Response Ratio

Summary:

The best way to guarantee success and generate high quality data is to first develop a stable and chromatographically correct HPLC method which resolves apart all of the compounds AND elutes everything off the column during the run. Do not start the calibration or quantification process until the method has been demonstrated to be stable, reliable and working perfectly. Problems seen with calibration methods are often caused by poor quality methods. Invest the time needed to produce an excellent quality method first and then you should have few to no problems later on.

*Related Reading: "External vs. Internal Standard Calibration in HPLC"; 

HPLC Column Support Pore VOLUME

If an HPLC column had no packing material inside it, then the volume of liquid contained in the cylinder could be calculated using the formula for the volume of a cylinder as follows: 

      Volume of Cylinder = Pi * r² * L;     

          [where Volume is in ul; Pi = 3.14; r = column radius (mm) and L= column length (mm)]

  Example: Using the above formula, a 4.6 mm x 250 mm column would have an empty volume of 4,155 ul (~ 4.16 mls).

For most chromatography applications we pack the column with a high surface area porous media. Often this is a silica based support. This support media fills the empty space inside the column reducing the total volume accessible by a liquid (or to the samples). If the media used was not porous, it would fill most of the space (depends on size and shape of media). Most commonly used chromatography supports are porous and leave about 70% (0.7) of the original volume available to the mobile phase and sample [Pore Volume = Surface Area (m²/g) x Pore Diameter  (Å) / 40,000]. Based on this information, we use a value of 0.7 as the average pore volume for a packed chromatography column (some supports will have pore volumes which are larger or smaller than this value. The manufacturer will often measure it and provide the value on their published specification sheet).


Using a typical 4.6mm x 250mm column we found the total volume to be 4,155 ul (4.16 mLs). If we now multiply this empty column volume by 0.7 (note: use 0.7 or 70% for columns with fully porous particles and 0.55 or 55% for superficially porous particles) we obtain 2,908ul total volume (2.9 mLs). This is the estimated volume of the fully packed column. This value is very important as it provides an estimate of what the column dead volume will be so we can calculate the 'T' zero time of an unretained analyte. This estimate will depend on the column dimensions, using our HPLC method (be sure and take into account the measured flow rate to determine the column "dead time"). This is one of the very first calculations you make when starting or modifying an HPLC method and is critical information to know at all stages of method development. All chromatographers should know how to estimate this value before using an HPLC system. *You should confirm this estimate by injecting an unretained sample onto the column and measure the retention volume, then compare the two values. The measured value is the most important number (the one we use for calculations), but the estimate should be close (+/- 15%). The estimate is still useful for troubelshooting and method development as when combined with K prime, it provides a quick measure if chromatography has occurred (retention).

For more information on the importance of knowing the HPLC Column Dead Time, please refer to this article link. 

Notes: The measured support pore Diameter (SIZE) is important for determining if the sample will have access to the inside of the support (e.g. A support with a pore size of 80Å will be too small for most large peptides or proteins, but a support that is 300Å will allow access to many, not all, larger molecules). A support with too small a pore diameter will not allow the sample to access the high surface area inside the support. Instead, the sample will be unretained and pass by it eluting at the column's void volume. This is the basis of SEC or GPC analysis where we use columns with different pore sizes to "filter" samples based on size. Large pores for large Mw samples and small pores for low Mw samples. A general rule is use 300Å or larger pores for samples with Mw > 10,000 and 80Å to 150Å for smaller samples.

More info on pore volume can be found at this article link: https://hplctips.blogspot.com/2014/12/hplc-column-pore-volume-or-pore.html

Common pKa Values for ACIDS & BASES used in HPLC and LC/MS Method Development

pKa (25°C)                              ACID

0.3                                           Trifluoroacetic acid

2.15                                          Phosphoric acid (pK#1)

3.13                                          Citric acid (pK#1)

3.75                                          Formic acid

4.76                                          Acetic acid

4.76                                          Citric acid (pK#2)

4.86                                          Propionic acid

6.35                                          Carbonic acid (pK#1)

6.40                                          Citric acid (pK#3)

7.20                                          Phosphoric acid (pK#2)

8.06                                          Tris

9.23                                          Boric acid

9.25                                          Ammonia

9.78                                          Glycine (pK#2)

10.33                                        Carbonic acid (pK#2)

10.72                                        Triethylamine

11.27                                        Pyrrolidine

12.33                                        Phosphoric acid (pK#3)

Notes: (1) This is a general list of commonly used acids & bases for chromatography applications and not meant to be a comprehensive list of all values. (2) TFA is an overused and very strong acid for many chromatography applications. It also has strong ion pairing properties and can result in high UV noise, vacuum degasser and/or MS contamination. If you must use it, try and use the lowest concentration which results in the desired pH. Example: 0.1 % TFA ~ pH 2.0, 0.02% TFA ~ pH 2.7. (3) Formic acid is a popular alternative to TFA for many applications, esp LC/MS. (4) Not all acids/bases provide "buffering" on their own.

Reference: CRC Handbook of Chemistry & Physics.