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1. What is the biggest difference between current HPLC phases and TYPE-C™ columns?



2. Buffered analytical eluents and Acidified solvents using phosphoric acid are suggested storage solvents for TYPE-C™ Columns, won’t they precipitate in the column?



3. How do I use these columns in Normal Phase after I have used them in Reverse Phase?



4. How do I condition TYPE-C™ Columns for use?



5. What is Silica-Hydride?



6. What Aqueous Normal Phase Chromatography?



7. Many other column suppliers suggest different columns for Acids, Neutrals or Bases, which TYPE-C™ column is best for each of these compound groups?



8. Cholesterol is a large molecule, is it stable as a stationary phase?



9. Why should I use a TYPE-C™ column to develop methods when I already have experience with Type-B columns?



10. Is TYPE-C Silica™ high purity and low metal content silica?



11. Will TYPE-C Silica™ have the same physical properties as my HPLC column does now?



12. What are the advantages of Silicon-Carbon bonding for stationary phases? How will that benefit me?



13. What is on-column hydrolysis and what causes it?



14. Is TYPE-C Silica™ a hybrid particle?



15. If I want to do Normal Phase with these columns, do I still use Phosphoric Acid in the solvents?



16. Do I use Phosphoric Acid if I scale up to preparative work?



17. I used a TYPE-C™ HPLC Column and dissolved my samples in Acetonitrile and water. Can I use other solvents such as methanol, IPA or Ethyl Acetate?



18. I am working with LC/MS, can I substitute Formic Acid for Phosphoric Acid with Cogent TYPE-C™ columns?



19. Which is better with TYPE-C Silica™ based HPLC Columns: TFA, Phosphoric Acid or Formic Acid?



20. What is the "Batch Reproducibility" of the Cogent Bidentate C18™?



21. Since you manufacture the Cogent Type-C™ silica using a transition metal catalyst, what if any is the presence of any trace quantity on the surface and how did you measure this?



22. What are the main differences between HILIC columns and a Cogent TYPE-C™ silica based column?



23. What is the concentrations of the test analytes in the actual test chromatogram that comes with the column?



24. Can I use Isopropyl alcohol as solvent in the mobile phase?



25. I am working with plasma extracts and we are diligent to remove our guard columns regularly but we would still like to perform a rigorous column washing to minimize contamination and carry over from run to run. What can you suggest other than extra column volumes of the mobile phase?



26. What other types of solvents can you suggest for rigorous column washing to remove adsorbed materials?



27. Most acids and bases are not effective when the organic content of the mobile phase goes too high. Since I am interested to use the Cogent columns in ANP, what can I do to adequately "acidify" the mobile phase when organic is higher than 40%?
    
    
28. How do I use Methyl Phosphonic Acid in my mobile phase?
    
    
29. Is Methyl Phosphonic Acid compatible with UV and MS detectors?

1. What is the biggest difference between current HPLC phases and TYPE-C™ columns? [top]
Over 95% of the surface silanols of our low metals, high purity silica has been replaced with silicon-hydride (Si-H) producing a stationary phase that adsorbs and de-sorbs mobile phase very differently than your current silica based phases. This key difference in our TYPE-C™ based columns provides many benefits such as retention of polar compounds that normally will not retain; retaining polar and non polar compounds in the same isocratic run; performing very rapid gradients; performing “reverse gradients”; performing organic normal phase with bonded phases such as C18 without drying out solvents; excellent peak shapes without end-capping. For more information on the differences, please click here.

2. Why use buffers during analysis and acid buffers such as the phosphoric acid for column storage solvents for TYPE-C™ Columns? will phosphate precipitate in the column? [top]
The TYPE-C™ phases can be stored in un-buffered aqueous / organic solvents, but flowing significant bed volumes of un-buffered aqueous will cause a drop in efficiency of approximately 35% before reaching a new steady state. This is why we recommend either acid or basic buffers but not un-buffered eluents during analysis.

For storage 0.04% v/v phosphoric acid ( 85% bench phosphoric acid ) is preferred.

No, Phosphoric Acid will not precipitate in the columns.

Sodium phosphate (which is not recommended) but which is often used as a buffer, will precipitate in the columns and should be avoided as a storage solvent for all HPLC Columns.

Phosphoric acid may be used to acidify 99+ % organic solvents such as methanol and acetonitrile without precipitation provided no sodium salt used.

3. How do I use these columns in Normal Phase after I have used them in Reverse? [top]
To convert the column from Normal Phase to Reverse Phase purge it with 15 column volumes of 100% HPLC Grade, filtered, Isopropanol (IPA). Be careful that the back pressure does not become excessive when purging with IPA as this is a more viscous solvent which can generate high back pressures even at low flow rates.

4. How do I condition TYPE-C™ Columns for use? [top]
If you are not converting to Normal phase, all you need to do is run 6 bed volumes through the column and then it is best to inject a known standard and repeat the injections until duplicate chromatograms are achieved. You should be able to achieve this within 2 to 4 injections.

5. What is a Silica-Hydride? [top]
This is our term for the surface of TYPE-C Silica™ which is dominated by silicon-hydride groups (Si-H). We are referring to the Silica Surface and the Hydrides.

6. What is Aqueous Normal Phase (ANP)? [top]
Aqueous Normal Phase (ANP) is a full, new mode of HPLC. Reverse Phase (RP) and Normal Phase (NP) HPLC are more well known but ANP offers chromatographers benefits that are not possible with other modes.

In Aqueous-Normal Phase, the maximum retention time of target compounds is with 100% acetonitrile (least polar solvent) and as you increase the polar solvent content (Aqueous), the retention reduces to a minimum when the mobile phase is at 70% Acetonitrile. You get Normal Phase mode using Aqueous or Reverse Phase Solvents. Polar compounds that would not retain in RP for example, retain very well in ANP.

By definition:
Normal-Phase Chromatography (NP)is a chromatographic mode that will increase the retention of a target compound as the mobile phase becomes less polar (a decrease in concentration of the most polar solvent, often water) conversely the compound has longest retention in a non-polar solvent such as 100% hexane. The main separation mechanism is based on the functionality of the analytes.

Reverse-Phase Chromatography (RP) is the opposite or the reverse of the above; there is an increase in retention time of the target compounds as the mobile phase becomes more polar (increase concentration of the most polar solvent, often water). The main separation mechanisms of RP is based on the hydrophobicity of the analytes.

Aqueous Normal-Phase Chromatography (ANP) is defined as a normal phase separation pattern using the reverse phase solvents Water and Acetonitrile. The main separation mechanisms of ANP is based on the functionality of the analytes.

7. Many other column suppliers suggest different columns for Acids, Neutrals or Bases, which TYPE-C™ column is best for each of these compound groups? [top]
The TYPE-C™ HPLC phases are unique in that they have different retention mechanisms which can be exploited to give unique separations. It may not be necessary to use different columns for different classes of compounds.

8. Cholesterol is a large molecule, is it stable as a stationary phase? [top]
Yes, the UDC Cholesterol phase has proven to be stable in 0.1% v/v TFA and 0.1% v/v H₃PO₄ at acid pH’s and up through pH 8.00 with 20mM NH₄COOH buffer with NH₄OH.

A pH in excess of pH 8.00 should be avoided as this phase has a mono dentate (single point) attachment to the silica matrix and an intermediate ester link in its structure. For higher pH’s the Cogent Bidentate C18™ is recommended.

9. Why should I use a TYPE-C™ column to develop methods when I already have experience with Type-B columns? [top]
Change is often work but can be worth every effort. With TYPE-C™ based columns, you can get good reverse phase from them but within the same solvent system (aqueous) you can achieve normal phase retention and elution patterns for polar compounds. You can also get polar and non polar compounds to retain and elute in the same isocratic run.

If gradients are required to cope with large differences in polarity of multiple compounds in a mixture the time it takes to equilibrate the TYPE-C columns is almost negligible. This makes your method development very fast. Only TYPE-C columns have Selectivity3™ which means on the same column, you can do Reverse Phase, Normal Phase and Aqueous Normal Phase.

Expand your skill set with TYPE-C based columns.

10. Is TYPE-C Silica™ high purity and low metal content silica? [top]
Yes, it is and it has all the advantages of high purity, type-b silica supports.

11. Will TYPE-C Silica™ have the same physical properties as my HPLC column does now? [top]
Yes, it will have all the mechanical strength, and durability that your modern, type-b packed HPLC column will have but will exhibit other very beneficial properties such as pH and temperature stability that is superior to your column.

12. What are the advantages of Silicon-Carbon bonding for stationary phases? How will that benefit me? [top]
Typical HPLC phases today are made with siloxane (Si-O-Si-C) bonds which are prone to hydrolysis. Silicon-Carbon bonds are much more stable and resistant to hydrolysis and is therefore less phase bleed-off. This means that these columns will last longer and be more rugged and will be reproducible longer. This also means that you might be able to explore new mobile phase/additive and temperature conditions to achieve very difficult separations that might be impossible with your current HPLC columns.

Also, standard type B columns made with silanization of organo-silanes to produce siloxane bonded ligands limits the phases which may be bonded onto silica. With our patented, silica-hydride surface many compounds previous thought to be improbable to bond to silica can be easily bonded. Examples of this is our unique UDC-Cholesterol™, plus our unique ability to custom synthesis novel phases for our customers.

13. What is on-column hydrolysis and what causes it? What will be the effect on TYPE-C™ Silica phases? [top]
Hydrolysis is a common degradation mechanism, which is facilitated by the presence of acids or bases in solution or on the surface of solids the liquid is in contact with. With solids in contact with liquids, the larger the surface area the greater the potential for hydrolysis. Irregular, type A & type-B HPLC phases all have by design very high surface to volume ratios plus Si-OH groups populate this surface. Even a fully end capped traditional phase can have 30 to 50% free silanols on its surface. Using photodiode array detectors and HPLC/Mass Spec has highlighted this degradation activity.

Certain analytical disciplines such as Natural Product Bio-actives and Forensic Science have shown that 10 to 20% of bio-active compounds may hydrolyze quantitatively “on-column” due to the conditions inside the HPLC Column. Historic analyses were usually made by these disciplines, of degradation products and not the injected material due to this activity in the column. TYPE-C Silica™ based products with silica-hydride surface (Si-H) are much less hydrolytically active than the historic silanol (Si-OH) populated phases and may not hydrolyze certain compounds making it possible to detect and track injected quantities instead of the degradation products inside created in the column.

14. Is TYPE-C Silica™ a hybrid particle? [top]
No, it is an evolution of standard, high purity, based deactivated, type-B silica and if you are using a commercially available silica based HPLC column, TYPE-C™ columns can be considered the same as what you are currently using.

15. If I want to do Normal Phase with these columns, do I still use Phosphoric Acid in the solvents? [top]
Usually it would not be necessary to use pH selectivity (the reason for adding phosphoric acid) with organic normal phase solvents, but this option is always option open to you for selectivity reasons. If chosen, it would be typical to use an organic acid or base rather than inorganic additives.

16. Do I use Phosphoric Acid if I scale up to preparative work? [top]
With preparative, reverse phase applications with TYPE-C™ columns, it is always preferable to use un-buffered eluents to reduce complications on evaporation. With TYPE-C™ columns significant volumes of non buffered aqueous/organic will cause a moderate but acceptable loss of efficiency. It is therefore recommended to use weak volatile buffers such as acetic acid/ammonium acetate/ammonium hydroxide mixes or their formate equivalents as these are easily removed during evaporation. If this proves to be a problem for your method, un-buffered solvents may be used, but the moderate loss of column efficiency will need to be allowed for and expected.

In most preparative applications reverse phase is rarely the first choice, as it is much easier to evaporate organic solvents than water-based solvents. Organic normal phase (ONP) is usually the best choice to start with.

TYPE-C™ columns which can work in aqueous normal phase (ANP) also reduces the water content more easily on evaporation, is therefore also a good choice for preparative work. In ONP it is rarely required to buffer the eluent and the TYPE-C™ phases is stable to all un-buffered organic HPLC solvents tested to date.

17. I used a TYPE-C™ HPLC Column and dissolved my samples in Acetonitrile and water. Can I use other solvents such as methanol, IPA or Ethyl Acetate? [top]
We do not advise TYPE-C™ users to use IPA for an eluent or as a solvent for target compounds. Regarding other solvents, it is always best to try to dissolve your target in as close as possible to your isocratic eluent or the start of your chosen gradient. If this is not possible gradually increase the percentage of the solvent in your eluent that the target is most soluble in.

18.I am working with LC/MS, can I substitute Formic Acid for Phosphoric Acid with Cogent TYPE-C™ columns? [top]
Yes, but monitor the pH. Keep it at approximately 2.00, no lower than 1.8 is recommended. Use approximately 0.05% Formic or 0.1% TFA (Trifluoroacteic acid).

19.Which is better with TYPE-C Silica™ based HPLC Columns: TFA, Phosphoric Acid or Formic Acid? [top]
TFA will usually provide better peak shapes than Phosphoric or Formic Acid, but Phosphoric is normally recommended for UV detector use since it is less hazardous than TFA.

TFA can cause some detector response - peak suppression with Mass Spec, but most users find this acceptable. If they cannot, then Formic Acid can be used, but we recommend using a combination of Ammonium Formate 20mM with Formic Acid buffered to approximately pH 2.2.

20.What is the "Batch Reproducibility" of the Cogent Bidentate C18™? [top]
For 8 consecutive batches of Bidentate C18™, carbon loading (excellent indicator for HPLC), did not vary more than 0.89%. This is almost within the experimental error of carbon load testing. Click here for more information.

21.Since you manufacture the Cogent Type-C™ silica using a transition metal catalyst, what if any is the presence of any trace quantity on the surface and how did you measure this? [top]
This is an issue we confronted a long time ago. We were also concerned about this potential problem. First we intentionally ran the bonding reaction at high catalyst concentration in order to actually reduce some of the Pt and deposit it on the surface. This produced a visual confirmation, the surface became gray. Next we ran some compounds that we knew would interact strongly with Pt and were able to observe longer retention and most important very distinct peak tailing. Finally we determined the %Pt on the surface by ESCA spectroscopy and it was close to 1 atom %. Then we went ahead and did our normal process. Of course the surface is very white under those circumstances. The compounds gave very good retention and peak symmetry was virtually 1.0. Then we took the material and analyzed it by ESCA. There was no Pt detected on the surface. The limit of detection is about 0.01 atom %. So we feel very confident that there is virtually no Pt present as determined by both ESCA and chromatographic testing.

22.What are the main differences between HILIC columns and a Cogent TYPE-C™ silica based column? [top]
There are many differences between HILIC columns and Cogent TYPE-C™ columns but for the sake of brevity, I will only answer with the main differences. The Cogent TYPE-C columns all perform similarly to HILIC as far as polar compound elution order is concerned when using higher than 70% organic composition of the mobile phase. Both columns perform separations that are based on variations of Normal phase called “Aqueous Normal Phase”. The HILIC stationary phase is typically more polar then the TYPE-C bonded phase which is relatively non polar. HILIC columns will retain polar compounds that cannot be retained by other columns and so will the TYPE-C columns. The HILIC columns will not retain non-polar compounds. This is one of the big advantages for the TYPE-C columns, where you will be able to separate polar and non-polar compounds in the same isocratic run.

On HILIC columns, polar compounds partition into and out of the hydration shell created by adsorbed water on the silica surface. As the ACN concentration increases the water layer decreases and the charged polar analytes are retained by the combination of cation exchange with the silanols under the water layer and the partitioning effect. The combination of these two mechanisms retains polar compounds in HILIC mode.

On Cogent TYPE-C Columns, the charged polar compounds elute in a similar order as on HILIC columns as stated above. However, non-polar compounds will be retained at the same time by the non polar ligand of the Cogent TYPE-C columns which is the bonded phase. Since there are virtually no silanols to speak of on these columns, the polar compounds are retained more by the adsorptive character of the silica-hydride and silica backbone which are now more available to the analytes due to a very shallow solvent layer which is the organic component such as Acetonitrile, Methanol etc. when at higher concentrations of the organic component.. Because of the lack of a “hydration shell” at high organic content due the weak association with water of the TYPE-C silica, the shell will equilibrate and change more rapidly with TYPE-C columns then with HILIC columns. This is an advantage for rapid gradients.

HILIC columns can only perform HILIC which is Aqueous Normal Phase while TYPE-C Silica based columns can perform Aqueous Normal Phase, Reverse Phase and traditional Normal Phase using completely non-polar solvents such as Hexane. There is no hysteresis when changing between these modes making this column extremely efficient.

23.What is the concentrations of the test analytes in the actual test chromatogram that comes with the column? [top]
It depends on the column and for the sake of brevity let me state the rationale we use to make up our standard test mixture for the Cogent TYPE-C columns.

Starting with stock solutions and toluene, we make up a 10ml final working solution from which we inject. The formula for making the final working solutions is as follows:

1. Uracil Stock Solution
   1. 1mg/ml dissolved in mobile phase
   2. 100ul into 10ml of the final working solution (mobile phase)
2. Anisole Stock Solution
   1. 200ul of Anisole dissolved in 20ml of mobile phase
   2. 800ul into 10ml of the final working solution
3. Toluene-Straight
   1. 30ul of Toluene into 10ml of the final working solution

24.Can I use Isopropyl alcohol as solvent in the mobile phase? [top]
We do not suggest using IPA (isopropyl alcohol) as component to your mobile due to the high back pressure you get from the high viscosity. However, it will not harm the column and many people use IPA as a driving solvent when switching from RP to NP and back and forth since it is miscible with both types of solvents. However, you should be extra careful to remove all of it from your column before using it for analysis.

25.I am working with plasma extracts and we are diligent to remove our guard columns regularly but we would still like to perform a rigorous column washing to minimize contamination and carry over from run to run. What can you suggest other than extra column volumes of the mobile phase. [top]
When working with plasma extracts, we recommend using 100% Acetonitrile for washing between injections. Flow rates of 0.5ml/min for 20-30 minutes for a 4.6mm x 250mm column should be sufficient.

26.What other types of solvents can you suggest for rigorous column washing to remove adsorbed materials? [top]
The Cogent TYPE-C columns are very stable and can be washed between runs with 100% Acetonitrile, DMSO THF and DMF among many others. We use these solvents when typical washing procedures are not adequate. The only pre caution I would give you is the stay between pH 2 and pH 10 when using acids and bases.

27.Most acids and bases are not effective when the organic content of the mobile phase goes too high. Since I am interested to use the Cogent columns in ANP, what can I do to adequately "acidify" the mobile phase when organic is higher than 40%? [top]
We have had some good success with methyl phosphonic acid. It remains soluble in acetonitrile content as high as 96%. This might help when some compounds are retaining in ANP but the peak shape needs to be tightened up a bit.

28.How do I use Methyl Phosphonic Acid in my mobile phase? [top]
You could add 1ml/L into the mobile phase. Add more or less as desired.

29.Is Methyl Phosphonic Acid compatible with UV and MS detectors? [top]
Methyl Phosphonic Acid, when used in dilute conditions as described above, is UV transparent and is volatile. Therefore you can use it with UV detectors. You should check with your Mass Spectrometer vendor to check compatibility as different MS instruments have different tolerances.