Preparation of calibration standards

Calibration standard preparation using volumetric flasks.

Published 6th March 2017 by Andy Connelly. Last updated 9th May 2017.


Calibration standards are key to the analytical process. They are used to create the calibration curve and the better the calibration curve, the better the data. In this blog post I will show various simple methods for making calibration standards from aqueous and solid standard materials. See end of post for a glossary of meanings.

DISCLAIMER: I am not an expert on analytical chemistry. The content of this blog is what I have discovered through my efforts to understand the subject. I have done my best to make the information here in as accurate as possible. If you spot any errors or admissions, or have any comments, please let me know.

The process

The process of making a calibration standards generally follows the process shown in Figure 1. The first two steps are covered in a previous blog post.

Figure 1: How to prepare calibration standard solutions.

Choice of standard material

There are lots of choices of standard material available, they can single element aqueous standards (e.g. 1000ppm Fe) or multi element aqueous standards. It is also possible to use high purity solid materials to prepare calibration standard solutions (e.g. using NaCO3 to prepare a 100ppm Na standard solution). The choice you make will be based on application, cost, availability, and the accuracy/precision you require.

Whichever you choose, it is important to use reagents suitable for the purpose. This will normally be analytical reagent grade chemicals (e.g. Aristar, Analar or equivalent) and ultra clean (Type I type) water. You will also need to decide on the composition of dilution (matrix) solution. For example, do you need to matrix match? Do you need to add acid (e.g. 1% HCl) to keep metals in solution, or base for iodine, etc? You may also need to add an internal standard to this solution for some analytical techniques (e.g. ICP-MS).


Before carrying out the calculation you need to decide on the concentration range of calibration standards and ensure the range covers the complete concentration range of your samples. In general, avoid serial dilutions if possible as these can propagate errors through standards which are hard to trace in a calibration curve.

To calculate the amount of aqueous standard material required to prepare a solution you can use the following equation:cvcv

where C = Concentration (ppm, kmol m−3, mol dm−3, etc.) and V = Volume (ml, dm, etc.)

As an example, to make 50ml of 5ppm calibration solution from a 100ppm standard (or stock) solution:



So, you would need 2.5ml of standard (or stock) solution and 47.5 ml of water to prepare 50ml of 5ppm calibration solution.

I have not covered calculations for the preparation of calibration standard solutions from solid standards. For more information on this please see, for example, here.

Preparing a stock solution

A stock solution acts as an intermediate solution between a standard solution and your calibration standards. For example, if you are calibrating in the range 1-10ppm it will be difficult to measure out the very small amounts of 1000ppm standard required. To make life easier you will generally make a 100ppm stock solution first and then use that to prepare the calibration standards.

It is not recommended to store any standard or stock solutions. However, more concentrated solutions generally store better as if a small amount of analyte sticks to walls of container it will not significantly affect overall concentration. As such, stock solutions can often be stored longer than low concentration calibration solutions. That said, storage of solutions is very analyte dependent (both in time and conditions) and should be avoided if possible.

Choice of vessel

Either volumetric flasks or centrifuge type tubes can be used to prepare a stock solution. Each vessel has advantages and disadvantages:

  • Glass volumetric flasks: larger volumes reduce the effect of measurement error and allow storage for future use. Use of glass is much better for organic analytes as not plastic. However, risk of contamination from glass (e.g. Na, K, Si, etc.) and analyte may stick to glass surface. If this a problem can use plastic volumetric flasks.
  • Plastic centrifuge tubes: you only make as much as you need so solutions are fresh, less risk of contamination as fewer processes. However, generally inappropriate for organics analysis, analyte may stick to plastic surface, also, requires pipetting of very small volumes so greater effect of measurement errors.

Whichever you choose it is important to use clean Class “A” glassware and calibrated pipettes. It is also good practice to check the calibration of the glassware, pipettes, and balances. This is especially true if you use variable pipettes.

If possible you should try to have all solutions, plasticware, and glassware at same temperature and otherwise you can have volume errors. Also, avoid over handling of equipment (e.g. volumetric flasks, pipettes, etc.) as this can cause warming.

Making a stock solution from a standard solution using a volumetric flask

All solution containers should be carefully labelled with your name, date of production and what is in the solution and the concentration.

  1. Decide on stock solution concentration and calculate volume of standard solution required (see above)
  2. Pre-rinse a volumetric flask (of appropriate volume) 3 times using ultra clean water. Keep stoppers on flasks except when adding water or reagents. Never put stoppers ‘nose’ down on the table (Figure 2 – A).
  3. Add ultra clean water (or matrix solution) into the volumetric flask (approx. 2/3 full). This can help reduce analyte ‘sticking’ to glass surface (Figure 2 – B).
  4. Add standard solution to a clean small plastic or glass beaker (e.g. medicine cup). Never put pipettes directly into the standard or stock solution container.
  5. Pre-wet the pipette tip 3 times with the standard solution from the beaker.
  6. Pipette appropriate volume of standard solution into volumetric flask being careful not to contaminate pipette tip (Figure 2 – C).
  7. Add ultra clean water (or matrix solution) until just below the meniscus line. Use a dropping pipette to make up to volume: bottom of the meniscus of the solution should be on meniscus line (Figure 2 – D).
  8. Put cap on volumetric flask and then invert and rotate 10-12 times to thoroughly mix solution. Ensure bubble in neck is completely displaced each time (Figure 2 – E).
  9. Use stock solution immediately or transfer to a clean and labelled reagent bottle (avoid storing solution in volumetric flasks).
  10. Discharge any remaining standard solution as appropriated. Never put it back into the bottle.
Calibration standard preparation using volumetric flasks.
Figure 2: Calibration standard preparation using volumetric flasks. See text for details.


Making a stock solution from a standard solution using centrifuge tube

As above except (see Figure 3):

  • Step 7: Instead of filling to meniscus line use mechanical pipette to fill to desired level. For example, if 2.5ml of stock solution required then pipette 12.5ml ultra clean water (or matrix solution) to make 15ml of stock standard. Some of water can be added before standard solution to avoid sticking.
Calibration standard preparation using centrifuge tubes.
Figure 3: Calibration standard preparation using centrifuge tubes. See text for details.

Making stock solutions from a solid reagent

As above except:

  • Check the percentage of desired analyte in solid reagent – remember to check water content.
  • Make appropriate calculations to prepare a stock solution from solid material. Avoid making all calibration solutions directly from solid powder if possible – make a stock solution.
  • If necessary, dry the solid reagent on a clean, oven dried, watch glass at 105 ºC for 2 hours and cool it in a desiccator. Check physical properties of reagent before this (e.g. melting temperature)
  • Weight the reagent in a tared clean weighting boat.
  • Carefully transfer the weighed chemical to a funnel placed on a volumetric flask. Wash the weighting boat with small portions of ultra clean water

Making standards

Whether you are going directly from an aqueous standard solution or from stock solution the method of preparation of calibration standards is very similar to the method for preparation of a stock solution.

Making calibration solutions from a stock solution

As above except (see Figures 2 & 3):

  • Decide on number (min. 5) and concentration of calibration standards and calculate volume of stock solution required for each. Repeat the protocol as many times as you require.
  • Decide on, and prepare, dilution solution (e.g. matrix solution, 1% acid, etc.) and use instead of ultra clean water as appropriate.
  • Step 4: can pre-rinse beaker with stock solution to clean.
  • Step 9: If using centrifuge tubes can use calibration solution direct from tube.
  • Avoid storage of calibration solutions.


The process for making calibration solutions is relatively simple. However, if you don’t take your time you can easily get things wrong. It is very frustrating to prepare your calibration standards, run them, only to find out that you have made a mistake and need to start again. Always remember, “Measure twice, cut pipette once!”

Further reading


  • Analyte: The component of a system to be analysed (e.g. nitrate, Fe, etc.).
  • Calibration standard: A dilute solution used in analysis to construct a calibration curve (e.g. 2,4,6,8,10ppm Fe)
  • Dilution solution: Solution you will use to dilute standard (or stock) solution to produce stock or calibration standards. Can be ultra clean water or another solution appropriate for your analysis (see below).
  • Matrix: The components of the sample other than the analyte (e.g. water, soil, etc.).
  • Matrix solution: A solution that matches matrix of sample (e.g. sea water, 10% acid solution, etc.).
  • Serial dilutions: calibration standards made by repeated sequentially dilutions using each new dilution as the “stock” solution. For example, diluting 1 ml to 10ml sequentially gives 1000ppm –> 100ppm –> 10ppm –> 1ppm.
  • Standard solution: A solution of accurately known concentration, prepared using standard substances. Often with a concentration of 1000ppm of analyte (e.g. 1000ppm Fe) and purchased with a certificate.
  • Stock solution: A concentrated solution that is used to prepare the calibration solutions. Generally of intermediate concentration (e.g. 100ppm Fe). You should always check this has been stored appropriately




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