1. What is the biggest difference between current HPLC phases and TYPE-C™?
   
   
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. Why should I use a TYPE-C™ column to develop methods when I already have experience with type-B columns?
   
   
9. Is TYPE-C Silica™ high purity and low metal content silica?
   
   
10. Will TYPE-C Silica™ have the same physical properties as my HPLC column does now?
    
    
11. What are the advantages of Silicon-Carbon bonding for stationary phases? How will that benefit me?
    
    
12. What is on-column hydrolysis and what causes it?
    
    
13. Is TYPE-C Silica™ a hybrid particle?
    
    
14. If I want to do Normal Phase with these columns, do I still use Phosphoric Acid in the solvents?
    
    
15. Do I use Phosphoric Acid if I scale up to preparative work?
    
    
16. 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?
    
    
17. I am working with LC/MS, can I substitute Formic Acid for Phosphoric Acid with Cogent TYPE-C™ columns?
    
    
18. Which is better with TYPE-C Silica™ based HPLC Columns: TFA, Phosphoric Acid or Formic Acid?
    
    
19. What is the concentrations of the test analytes in the actual test chromatogram that comes with the column?
    
    
20. 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%?
    
    
21. How do I use Methyl Phosphonic Acid in my mobile phase?
    
    
22. Is Methyl Phosphonic Acid compatible with UV and MS detectors?

1. What is the biggest difference between current HPLC phases and TYPE-C™? [top]
The biggest difference is the silica surface and how it will benefit the user. The main difference in the silica’s is the dominance of Silicon-Hydride (Si-H) groups on the TYPE-C™ products instead of the Silanol Group (Si-OH) common to all irregular, type A & B silica based previous phases. This difference in polarity and the novel separation mechanisms of TYPE-C™, gives new selectivity options for these HPLC columns as they can all be used in organic and aqueous normal or aqueous reversed phase even C18.

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 5 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? [top]
Normal-Phase Chromatography is defined as a chromatographic phase 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.

Reverse-Phase Chromatography is the opposite or the reverse; 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).

Aqueous Normal-Phase Chromatography will be defined as a normal phase separation pattern using the reverse phase solvents Water and Acetonitile.

In Aqueous-Normal Phase, the maximum retention time of target compounds is 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.

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. Click here for a Real Life Example of the Cogent UDC-Cholesterol™ column separating two very different types of compounds.

8. Why should I use a TYPE-C™ column to develop methods when I already have experience with Type-B columns? [top]
Often gradients are required to cope with large differences in polarity of multiple compounds in a mixture. With TYPE-C™ columns there always is the possibility to achieve isocratic analyses where traditional HPLC Columns could require gradients. As can be seen from a real life example TYPE-C™ columns also have the ability to retain compounds that cannot be retained on previous types of supports and phases.

Also, the lack of water retention on TYPE-C™ columns makes change of pH and switching between aqueous reverse/normal phase to organic normal phase rapid and memory free. TYPE-C™ columns have multiple separation mechanisms at work, which can be modified by choice of appropriate bonded phase to interact or act separately. Unique but predictable separations can be extrapolated from retention maps.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17.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).

18.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.

19.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

20.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.

21.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.

22.Is Methyl Phosphonic Acid compatible with UV and MS detectors? [top]
Yes, Methyl Phosphonic Acid, when used in dilute conditions as described above, is UV transparent and is volatile. Therefore you can use it with UV or MS detectors.