1. What is mixed-mode chromatography, and how is it different from reversed-phase, normal phase and HILIC approaches?

Mixed-mode chromatography is defined as separation technique where at least two controllable interactions exist in order to adjust selectivity and retention of compounds. Mixed-mode chromatography can combine reversed-phase or HILIC interaction with ion-exchange, ion-exclusion or pi-pi interaction.

2. Are mixed-mode columns compatible with 100%-aqueous and 100%-organic mobile phases?

Mixed-mode stationary phases have ionizable groups on the surface, allowing analysis with both aqueous and fully organic mobile phase. Polar groups on the surface prevent phase collapse that could occur in reversed-phase columns.

3. What kind of compounds can be separated and retained on reversed-phase mixed-mode columns?

Mixed-mode chromatography provides retention and separation for a much wider range of compounds than single-mode chromatography. You can retain and separate neutral hydrophobic, hydrophobic ionizable, polar, and polar ionizable compounds, along with positively and negatively charged inorganic ions, within one run.

4. What kind of compounds can be retained and separated on HILIC mixed-mode columns?

HILIC mixed-mode columns are the ideal choice for separation of polar neutral, polar ionizable, and hydrophobic ionizable compounds, and positively and negatively charged inorganic ions.

5. What kind of buffers and additives I can use in mobile phases?

You can use any ions or additives as long as you stay within the recommended pH range for specific columns. You need to make sure that your additives or buffers are compatible with your detection technique, and that your buffers are soluble within the operating ranges of your solvents.

6. What kind of gradients I can use in mixed-mode chromatography?

Since columns are compatible with 100%-organic and 100%-aqueous mobile phase, you can use any gradient. Just remember that some compounds are retained and separated by ion-exchange mechanism and that you may need to maintain ions in the mobile phase to facilitate elution and to obtain good peak shape.

7. Can I use ion-pairing reagent with mixed-mode columns?

You don’t need to use ion-pairing reagents to retain your compounds on mixed-mode columns. In reversed-phase mixed-mode columns, the ion-pairing reagents are attached to the surface of silica gel, thus making the use of ion-pairing reagents for retention of polar ionizable compounds unnecessary.

8. What are most popular column sizes people use in mixed-mode chromatography?

Mixed-mode columns have a much higher capacity than single-mode columns, with most common columns 50, 100 and 150-mm in length. Using shorter column allows you to develop shorter methods without sacrificing resolution between compounds.

9. What conditions should I keep in mind when choosing a column and mobile phase?

In order to choose the right column and mobile phase, you need to understand the properties of the stationary phases and your analytes. If you want to add an ion-exchange interaction to your HILIC or reversed-phase separation, you need to make sure that both the stationary phase and your analytes are ionized and available for ion-exchange or ion-exclusion interaction.

10. I need to isolate impurities in my API – how can I benefit from using mixed-mode columns?

Mixed-mode columns have a 5-50 times higher capacity towards ionizable analytes. This helps load more on the column and isolate large quantities than in single-mode chromatography. Presence of two interactions can help you to independently adjust retention time and to elute your impurities either before or after your peak, depending on your task.

11. Can I use alcohols with mixed-mode phases?

Some mixed-mode columns have carboxylic acids on the surface of silica gel, and under certain conditions you can esterify carboxylic groups on the surface and change selectivity of your separation. This may be very critical when your method is moving toward validation. Most of our mixed-mode columns use a different column chemistry, and can be used with alcohols without modifying the stationary phase.

12. Can I use alcohols as diluents for my analytes?

As long as your compounds are not reacting with alcohols you can use them as diluents without any problem.

13. What kind of gradients I can run?

You can run single, double and triple gradients. In your gradient you can modify the amount of organic component of mobile phase, amount of buffer, and buffer pH.

14. How long is equilibration time in mixed-mode chromatography?

If you stay with the same buffer, equilibration in mixed-mode chromatography is the same as in single-mode chromatography. If you change the nature of the buffer, you may need a longer time for equilibration and/or higher concentration of the buffer. Once the buffer is replaced, equilibration times are similar to single-mode chromatography.

15. How can I adjust selectivity of my separation if I observe co-elution of compounds?

You can use four parameters within the same column to adjust your selectivity: amount of organic, buffer pH, buffer concentration, and nature of the buffer. All these parameters can help you achieve the desired resolution and retention time. In many cases you can change order of elution for your compounds by changing the buffer pH and buffer concentration.

16. When I inject my compound into mixed-mode column I don’t see any peaks?

Make sure that the absence of peaks is not a result of malfunction of your equipment, and that your compound is visible with the specific detection technique you are using. Once you eliminate potential issue with equipment, you can start modifying your mobile-phase composition. In majority of the cases you need to increase strength of your mobile-phase by increasing (or decreasing) the amount of organic component of the mobile phase (in both HILIC and RP modes), and by increasing the amount of ions in the mobile phase. You also can adjust pH in order to decrease ion-exchange interaction between charged analytes and your mobile phase.

17. How can I choose what column to use?

You need to evaluate the hydrophobic/hydrophilic properties of your analyte and your column. Make sure that you don’t have two very strong interactions in order to avoid very long retention time or very high buffer concentrations.

18. I made injections into mixed-mode column and I see peak in the void – how can I increase retention time?

First make sure that whatever you see is your compound and not an impurity or baseline distortion caused by injection. You can inject your compound without a column by using bypass and calculating peak area. In majority of the cases, the sum of all peaks with the column should be very close to peak area of peak without the column. If you discover that the sum is the same, you need to decrease the strength of your mobile phase, or switch to a stronger mixed-mode column.

19. How do I increase/decrease strength of the mobile phase in reversed-phase mixed-mode chromatography?

For compounds that are retained by reversed-phase mechanisms, increasing or decreasing the strength of the mobile phase is done in the same way as in single-mode reversed-phase chromatography. More organic component of the mobile phase will facilitate elution, while less organic will keep your compounds longer in the column. For ionizable compounds you need to consider in what mode you operate – ion-exchange or ion-exclusion. In ion-exchange mode you increase the strength of the mobile phase by increasing buffer concentration, and reduce the strength of the mobile phase by using less ions. In ion-exclusion mode you increase ion-exclusion interaction by decreasing the amount of ions in the mobile phase. You can also increase or decrease the strength of the mobile phase and ion-exchange/ion-exclusion interactions by playing with pH, but in this case you need to consider both the ionization state of stationary phase and the ionization state of your analyte. For non-zwitterionic, but ionic, compounds, an increase in pH usually provides stronger interaction.

20. How can I improve the peak shape of my analytes?

Mixed-mode columns provide much better peak shape than single-mode columns. If your compound comes out with distortion or poor peak shape, most likely you are using a wrong column or wrong pH. You need to make sure that you are controlling ionization state of the column and stationary phase. Another reason for poor peak shape might be overloading of the column, which can be corrected by using a stronger mixed-mode column, using a weaker mobile phase, or injecting less.

21. What is pressure rating of your columns?

Our hardware is rated to 15,000 psi.

22. What is temperature rating for columns?

Most columns can be operated within 20 to 60 degrees Celsius.

23. Can I used THF in the mobile phase?

As long as your instrument can tolerate the use of tetrahydrofuran you can use it with our columns.

24. Where can I buy mixed-mode columns?

You can buy our columns directly by emailing us your purchase order or through our distributors. We usually ship within 1-3 days of receiving your purchase order.

26. How reproducible are your columns?

We keep our reproducibility within 3% between lots. If you observe a larger variation than that, most likely you are comparing an old column that was exposed to harsh conditions to a new column, or your mobile-phase conditions are different.

27. How accurate do I need to be with pH adjustment of the mobile phase?

We recommend you keep the pH within 0.05 units to ensure robustness of the method and reproducibility of the separation.

28. What particle sizes do you offer?

We offer 3, 5 and 10 um columns packed with regular spherical silica gel, and 2.7 um columns with core-shell mixed-mode columns.

29. What sizes of columns do you offer?

We offer 50, 100, 150 and 250 mm columns with 2.1, 3.0, 4.6, 10 and 21.2 mm diameters.

30. Can you help me developing a method?

We offer comprehensive method development screening, as well as individual and group training and seminars. You can contact us by email or phone to inquire about our services and seminars.

31. I am observing unusually high back-pressure on my columns – what should I do?

Assuming that you don’t have problem with your equipment we would recommend you change the tubing, and wash the column in opposite direction to make sure that there is no build-up at the top of the column.

32. If my column is contaminated, how can I clean it?

You need to use a stronger mobile phase with more ions and wash column in opposite direction. For reversed-phase mixed-mode columns use 60-70% of acetonitrile and 0.3% of sulfuric or phosphoric acid. You can use 50-100 mM ammonium formate buffer pH 3 with 60-70% of ACN. For HILIC mixed-mode columns use less acetonitrile (20-30%) but the same amount of additives as in RP column. Sometimes washing overnight with a reduced flow rate can solve contamination problem. If washing overnight does not address the issue of contamination, you will need a new column.

33. How many lots of silica gel you have in stock?

We keep at least two lots of each stationary phase in stock, more lots can be produced within two weeks to satisfy your validation requirements.

34. I got your column from a colleague, can I use it for method development?

We don’t recommend using columns from somebody else, as you don’t know how this column was used and stored. You can use the column for preliminary screening, but in case of method development and validation we recommend buying a new column.

35. What are lot numbers and serial numbers on the column?

The lot number represent the material that was used to pack the column. Serial number is a specific and unique number for a specific column. Columns of various length can be packed with the same material but each has a unique serial number.

36. I lost my Certificate of Analysis, how can I get a new one?

You can request a copy of COA from our customer service.

I’ll work on this (k, i’m done with 1-35, will come back later to recheck

37. How I should approach method development tasks?

The key to successful method development is understanding properties of stationary phase and properties of compounds. You need to know ionization states of compounds and stationary phases, and how buffer pH and buffer concentration affect interactions on the column. We recommend to start method development with a short 50-mm column to experiment with how compounds interact with the column. Once you learn the interactions you can move to a longer column if the resolution between peaks needs improvement. The easiest way is to modify only one parameter of the mobile phase at a time. Change the amount of organic in 10% increments and buffer additive in 10 mM increments. If your compound is neutral the most effective way to adjust retention is to change amount of organic component of the mobile phase. If your compound is hydrophilic and ionic, then the most effective way to change retention is to play with buffer concentration and buffer pH.

38. How do I prepare and adjust pH of the mobile phase?

We recommend to prepare aqueous solution of your buffer and then use the same acid as in your buffer to adjust pH – formic acid for ammonium formate, acetic acid for ammonium acetate. For more robust method please keep pH within 0.05 units of desired pH.

39. If my compound is not eluting with formic or acetic acid in the mobile phase what should I do?

You can use a corresponding buffer. Salts create more ions and can be used to increase the strength of the mobile phase.

40. What is the most effective way to set up HPLC system for method development with mixed-mode columns?

We recommend using quaternary pump for method development. Once you develop your separation you can switch to a binary pump. For quaternary pump, set up your channels as follows: channel A – water, channel B – acetonitrile, channel C – 100 or 200 mM buffer, channel D – 1 % acid (phosphoric, sulfuric, methanesulfonic, perchloric, TFA, formic, acetic). Use certain percent from buffer or acid channel to create desired concentration of ions. For example, if you set up channel C at 10%, you will be delivering 10 mM of buffer into the column (in an example of 100 mM stock solution of the buffer). Quaternary pumps help you avoid preparing numerous pre-mixed mobile phases, and can significantly increase efficiency of your method development.

41. How do I request method development screening?

If your company requires CDA or NDA, please email all legal forms you would like us to sign. Once you are free to share information about your compounds, please fill method development form with as much information as possible. We will review your requirements and provide you with an estimate how much it will cost and how much time do we need. We will require individual samples/markers (if possible) in the amount of 10 mg+. We do not have MS capabilities and cannot identify individual compounds unless we have standards or markers.

42. What if I don’t see my specific compound on your list of applications?

There are two approaches to get help: you can look at similar compounds which have same functional groups, or you can ask us to develop a method for you. Our philosophy is that you don’t need to know the exact structure of your compounds to develop a method. Functional groups on a molecule define what interactions you have on the column. If you know the interaction, and how to control them, you will be able to develop a method with minimum guidance from us, often using a similar applications as a starting point of your method development.

43. Are your columns compatible with LC/MS and prep chromatography?

Mixed-mode columns were designed to work with mass spec detection. You don’t need to use TFA or any other ion-pairing reagents. Retention for polar ionizable analytes comes from interaction with ion-pairing reagent attached to the surface of silica gel. You can use any volatile mobile phase with our columns as long as you stay within the recommended pH range.

44. What are common mistakes people are making when developing and validating a method on mixed-mode column?

The most common mistakes are:

  • Make sure that you know properties of your compounds.
  • Read column care instruction to know pressure, pH, and temperature limits of the column, as well as compatibility with different solvents.
  • Choose a detection technique where you can monitor your compounds. Use two detection techniques if possible to ensure the “visibility” of your compounds.
  • Make sure that your mobile phase is compatible with your detection technique and with the column.
  • Never use column from unknown source. Borrowing column from your colleagues might be dangerous since you don’t know history of the use and storage.
  • Choose the right dimensions of the column. Mixed-mode usually provide much longer retention so you might need a shorter column.
  • Use of much-higher-than-recommended flow rates usually means that you chose the wrong column or mobile phase.
  • Develop isocratic method if possible – your method will be shorter and more robust. Specifics of mixed-mode chemistry allows you to replace gradient method with isocratic.
  • If you plan to use ion-exchange or ion-exclusion mode in addition to HILIC or RP, make sure that your compounds and stationary phase are ionized.
  • Make sure that you are eluting everything from the column, and that whatever you see in a chromatogram are your compounds and not a carry over or some minor impurity in your mix. Injecting analytes with and without column can help you insure that your compounds are eluted from the column.
  • Make sure that you are equilibrating the column, particularly when you change nature of your buffer/additive. Mixed-mode columns has a huge capacity and you need to consider this when equilibrating the column. Make several injection to make sure that your retention time does not change from one injection to another.

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