Method Development Tips


Method Development Steps for HPLC Analysis of Various Compounds

Reproduction of Existing Method from SIELC

1. Review the method and make sure that you have exactly the same column as in the method description. Your column type and dimensions should be exactly the same as in the method which was communicated by us. If you column type or dimensions are different, then modification of method conditions are required and your separation can be greatly affected with possibility of no separation or retention.

2. Do not try to change ANY of the parameters of the method as we most likely already screened different conditions and obtained optimum results for your specific set of compounds.

3. Make sure that you have the same detection technique as described in the method. If your detection technique is different make sure that your detector is compatible with the mobile phase and with the method. If your detection is different make sure that your compounds can be detected by this detection technique.

4. Read column care instruction and understand limitations of the column in terms of solvent and buffer selection, buffer pH and temperature. Column care instructions can be downloaded from

5. Make sure that you are using new column if method development and validation are required. If you column is from an unknown source (used by your colleagues) be careful because you don’t know how the column was used previously.

6. If column is from an unknown source, use it only for screening and to learn how retention time is controlled on this specific column.

7. Prepare the mobile phase for the method. In all SIELC methods, if the description states X% of organic with Y mmol (or Z% of acid), that is the amount delivered into the column, usually by premixing in a bottle or premixed by pump.

8. If the mobile phase setup requires pH adjustment, use corresponding acid/base to adjust pH within 0.01 unit limit. If you are using ammonium formate, then formic acid is used for pH adjustment, if your buffer is sodium phosphate then either sodium hydroxide or phosphoric acid are used for pH adjustment. Make sure that your pH meter is calibrated before pH measurement.

9. If you are using quaternary pump, prepare stock solution of the buffer or acid which is 100 or 200 mmol (1 or 2% for acids). Use channel A for water, channel B for acetonitrile (methanol in some cases) and channel C for your stock solution of the buffer. Make sure that your stock buffer concentration can deliver required amount of the buffer or acid with the certain amount of organic solvent.

10. Use A, B and C channels to create desired compositions of the mobile phase. If method is calling for 20% ACN and 40 mmol buffer (or 0.4% of acid), set up your pump as follows: A-100% water, B-100% ACN, C-100 mmol buffer (or 1% of acid). In order to deliver desired composition you need to get 40% from channel A, 20% from channel B and 40% from channel C. Same principle is applied for gradient when you set up amount of acetonitrile and buffer and water channel is a “makeup channel”

11. If you have a binary pump, make the same buffer as above and premix mobile phase in channel A or channel B. The above mentioned mobile phase is created by mixing 200 ml of acetonitrile, 400 ml of buffer and 400 ml of water.

12. If you changed any of the bottles in your system make sure that you purge all lines with the corresponding component.

13. SIELC columns have huge capacity and if you don’t know how the column was used before or if it just came new, you need to replace the previous mobile phase buffer or shipping solvent (usually ACN/water/TFA in case of shipment). In order to shorten buffer replacement it is recommended to use a higher concentration of the buffer (50-80 mmol). Consult this table for equilibration times when one buffer is replaced with another:

14. After you have equilibrated the column, the method can be set up as described.

15. Prepare 1-2 mg/ml solutions of your samples in ACN/water. If the sample is not soluble try to add alcohol or a small amount of THF, DMF, DMSO. Make a note of how much solvent you added. You might need to prepare a solution of this additive in ACN/water to have a marker/reference point.

16. Do not prepare a mixture of compounds or analyze the mixture before finding out when each compound is eluting.

17. Inject your compounds without the column to make sure that your injector works properly and that you can observe compounds and measure peak area.

18. Inject in duplicates individual compounds with the column. Make sure that your compounds are eluting and that retention time, peak area are the same for both injections. If they are different, then something is wrong and you need to troubleshoot the issue.

19. If your method setup is correct you should be able to reproduce our method. If the method is not reproducible, then you either using a wrong column, an old column which was modified by another user, something is wrong with your HPLC system or something is wrong with your mobile phase.

20. Contact us with a detailed description on how you prepared your mobile phase and serial number of the column if your method is different than the one we communicated to you. Use for all communications related to methods and columns.


Method Development for Compounds with Unknown Structure or Properties

1. Collect data on UV activity of compounds to know if UV detection will work for your unknown compounds. If compounds are not UV active, consider using a different detection technique, like ELSD, CAD, MS, RI, conductivity or electrochemical. Collect as much supplemental data as possible (IR, NMR, UV, etc.). This data will allow you to make a preliminary judgement on chemical and physical properties of your compounds.

2. Decide which buffer or acid you are going to use. This will depend on what detection technique you are going to use. Your mobile phase additive must be compatible with your detection technique. Use this link to decide on the mobile phase selection:

3. Prepare a stock solution of your buffer or acid – make 100 mmol stock solution of salt buffer or 1% of the acid in water. This will be used to create desired mobile phases

4. If you have quaternary pump, set up channels as followed: A- water, B-ACN and C your stock solution of the buffer or acid. If you have binary pump, then you will need to pre-mix your mobile phases

5. Decide on the high and low values of ACN and buffer (or acid) concentration. We would suggest 20% and 60% for ACN. For acids, these values are 0.05% and 0.25%, for buffers, it is 10 mmol and 40 mmol. Make sure that your buffer is soluble at a higher organic concentration than you are using.

6. Read column care instruction to make sure that your mobile phase is compatible with the column

7. Prepare 1-2 mg/ml solutions of your samples in ACN/water. If the sample is not soluble try to add alcohol or small amount of THF, DMF, DMSO. Make a note of how much of the solvent you added. You might need to prepare a solution of this additive in ACN/water to have a marker/reference point.

8. Do not prepare a mixture of compounds or analyze the mixture before finding out where each compound is eluting.

9. Start with the method development kit consisting of Primesep SB and Primesep 200 column (50 mm length). The short column will allow you to do method development much faster. Once you understand how to control retention time for your unknown analytes, you can move to a longer column if better resolution is required and cannot be obtained on a short column.

10. If your detection technique is UV, set up several wavelengths (210, 230, 250 and 270)  and at least one alternative detection technique (ELSD, CAD, MS) to make sure that you can monitor. Make sure that your buffer is compatible with your detection. Cutoff for ammonium formate, ammonium acetate, formic and acetic acids is 230 nm.

11. Inject each compound without the column and measure peak area. Make sure that you see each compound and that you are not overloading the detector. This is your primary indication that you can most likely use the chosen detection technique.

– ACN: 60%, Acid 0.25% (in case of the buffer use 40 mmol)
– ACN: 20%, Acid 0.25% ((in case of the buffer use 40 mmol)
– ACN: 60%, Acid 0.05% (in case of the buffer use 10 mmol)
– ACN: 20%, Acid 0.05% (in case of the buffer use 10 mmol)

12. Equilibrate your column with 20% ACN and 0.2% acid (or 50 mmol buffer) for 20 minutes.

13. Set up a run time of at least 12 min on 50 mm column. Run the following experiments on Primesep 200 column for each individual sample, making 2 consecutive injections:

14. Compare peak areas without column and with the column. The sum of all peaks in your individual chromatogram with the column should match peak area without column. If there is mismatch of more than 20% you are not eluting everything you injected and this mobile phase is too weak for this specific compound

15. Assuming that you have eluted everything from the column for each of the individual compounds with any of these methods, compare data for 4 runs.

– If your compounds in runs B and D have longer retention than in run A and C they are hydrophobic in nature. If you don’t observe significant shift in retention, most likely your compound has low hydrophobicity
– If your compounds in runs C and D retain longer than in runs A and B, your compounds are most likely are basic in nature. Compounds which have lower retention in C and D and compare to A and B are most likely acidic and need to be analyzed on Primesep SB if retention is not significant on any of the conditions you screened
– Compounds which retain longer in run D compare to run A, B and C are most likely are both hydrophobic and basic in nature
– Compounds which did not respond to change in amount of ACN and ions are most likely neutral and hydrophilic, or they are not ionized at your mobile phase conditions

16. Once you learn the nature of your compounds you can further adjust your mobile phase to achieve separation. Retention of hydrophobic compounds is adjusted by amount of ACN -more ACN the shorter is retention, Retention of ionic compounds is adjusted by amount of ions in the mobile phase and pH of the mobile phase. More ions will elute your compounds faster in ion-exchange mode.

               If you are satisfied with retention you can stay with the chosen conditions, if retention is too short for compounds, you can further reduce amount of ACN from 20% to 10% and re-run experiments.

17. Once you run all your experiments, evaluate the data and see if running a gradient is an option.

               Set up your system as follows:
                    – Channel A: 10% ACN with 0.05% of acid (or 10 mmol buffer)
                    – Channel B:  60% ACN with 0.25% of acid (or 40 mmol buffer)
                    – Run gradient from 100% A to 100% B in 8 minutes with 4 minutes hold
                    – Total run time 18 minutes

18. Repeat all steps above with Primesep SB if you compounds are not retained or you are looking at alternative selectivity

19. Explore another pH of buffer

20. You can also explore a weaker or stronger column.

               Here is the relative strength or reversed-phase cation-exchange columns/applications:
                    – Primesep A>Primesep 100>Primesep 200>Primesep C>Obelisc R>Primesep 500
                    – For reversed-phase anion exchange columns/applications:
                    – Primesep SB>Primesep D>Primesep B2>Obelisc R

21. If none of your compounds are retained in reversed-phase ion-exchange, switch to HILIC-ion-exchange on Obelisc N Column

22. For HILIC ion-exchange on Obelisc N column conduct all steps as above with a different mobile phase composition.  You need to stay with pH 3-6, ACN concentration 50% and 85%, buffer concentration of 5 to 50 mmol

Method Development for Compounds with Known Properties or Structures

For compounds with known structures and properties use our Method Development chart, brochures, methods from our website. Newsletters in Literature section:

or our our interactive web method development tool: