HPLC, or high-performance liquid chromatography, has been around for more than 50 years. In that time, science has drastically improved the reliability and accessibility of this method.
But how does HPLC work? How do you know which column to use? Do you know the difference between what is C18 and C8?
Don’t worry, we’re here to help. Here’s our guide on the basics of HPLC columns, and this is our flagship C18 Column.
How Does HPLC Work?
HPLC is used to separate, quantify, or identify components in a mixture. The components are separated using column chromatography and then analyzed by a computer.
All forms of column chromatography work similarly. You pass your mobile phase (the liquid containing your mixture) through a stationary phase (a solid). The components will travel through the solid phase at different rates, which allows them to be separated over time.
In HPLC, high pressure is applied so that this separation can occur much more quickly than traditional means. Additionally, using smaller particles in the column’s packing material permits more precise separation.
HPLC has three steps:
1. A small volume of your sample (in the liquid phase) is injected into your stationary phase. Your stationary phase is your HPLC column, which is a tube filled with particles <5µm in diameter.
2. A pump moves the liquid down the column using high pressure. As the sample moves through the column, there are interactions between the packing particles and molecules in your sample. This will cause these molecules to travel through the column at different rates.
3. As the individual components exit the column, a detector measures them. This output is sent to a computer to produce a liquid chromatogram.
In HPLC, the column you use is often considered the most important component. The physical and chemical characteristics of the column determine the degree of separation. But how do you know what column is right for your experiment?
Determining Your HPLC Column
First, you’ll need to establish if you’re performing normal-phase or reversed-phase HPLC. In normal-phase HPLC, the stationary phase is hydrophilic while the mobile phase is hydrophobic. But reversed-phase HPLC is far more common.
In reversed-phase HPLC, the mobile phase is hydrophilic while the stationary phase is hydrophobic. That means that molecules are eluted by decreasing polarity through the use of an organic solvent.
Now you’re ready to pick out your column. But what features are the most important?
Base Packing Material
Silica gel is by far the most common base packing material.
The surface has silanol groups, which are highly polar and can interact with non-polar molecules in your liquid phase. They can also serve as chemical bonding sites. And the large surface area offers a strong adsorptive capacity.
Silica particles are rigid, which helps them resist compaction. This is crucial when you’re using extremely high pressures for very small molecules. Low acidity silica is used to avoid interactions between bases and your liquid phase, improving peak shape.
If you need a column that can work at extreme pHs, polymer packings are a solution if you’re working on a smaller scale. They are stable and do not leach the liquid phase. Additionally, a variety of coating options offer more functions for your column.
Pore and Particle Size
The pore size you want is based on the molecular weight of your compound.
The smallest molecules should be less than 120Å. Polypeptides and compounds with several proteins should be about 200-400Å. Very high molecular weight proteins should be 1,000-4,000Å.
Particle size used to always be the standard 5 microns. In the mid-90s, this standard lowered to 3.5 microns.
Now, if you want faster or better resolution, you can choose even smaller particles (even as little as <2microns). But note that for these, your columns will need to be shorter for standard equipment. A lot of this has to do with the increase in backpressure, which increases with both column length and decreased particle size.
While longer columns are possible, they can only be used by machines that operate at a higher pressure.
Again, the column dimension used to be standardized along with particle size (4.6 mm x 100 mm). If a higher resolution was required, 150 mm or 250 mm columns were used.
Smaller column dimension sizes are preferred because they are cheaper and use up less of your sample. If you have small amounts of sample, consider nanocolumns (<1 picogram), capillary columns (picograms to nanograms), or microbore columns (nanograms to micrograms)
Reducing diameter can also result in much greater sensitivity. For example, halving the diameter can quadruple the sensitivity.
The last aspect you’ll need to consider is the bonded phase. These are the chains attached to the surface of the silica. Common bonded phases include C18, C8, and phenyl. But what is C18, and how does it compare to the other types of bonded phases?
What is C18?
C18, C8, and C4 are all linear alkylsilane phases. C18 is octyldecylsilane and contains 18 carbons bound to the silica. So they have more carbons and a longer carbon chain than C8 (8 carbons) or C4 (4 carbons).
Because of the extra carbons, C18 has a larger surface area that the mobile phase has to travel across. This offers more interaction time between the bonded phase and the elutes. Thus the sample elutes more slowly and has more separation.
In contrast, C8 (also called octyl) is better for shorter retention time and provides sharper peaks. These column types are better for small organic compounds, while C18s are better for long-chain fatty acids and complex molecules.
Buy Your Perfect HPLC Column Today!
Now that you know what is C18 and all the other basic HPLC column questions, you’re ready to determine the HPLC column that works best for you. Want the very best for your experiment? Buy Develosil HPLC columns!
We offer a wide range of HPLC columns that are both durable and flexible. Contact us today to find out which of our high-quality columns are the right choice for your experiment.
Thanks for valuable info. I need more info on Synthetic Peptide Purification by RP HPLC.
Thank you for your comment. Here is a link to a recent app note for large size protein/antibody purification that may be of interest to you: https://develosil.us/wp-content/uploads/Antibody-Analysis-with-UHPLC-MS.pdf. Please let us know if you have any more questions at email@example.com and a representative will contact you.
Develosil US Team.
Thanks for valuable information
Hi and thank you for your comment. We are happy to hear you found our post valuable. Please feel free to contact us if you have any questions.
C18 is octadecyl but how can C8 be also octadecyl…..it should be octyl I think…
Thank you for pointing that out. You are correct, C8 is called an octyl. We have corrected the text.
Which one is better for compounds like karanjin which may behave polar and non polar both depending upon the eluting solvent?
Thank you for reaching out. We haven’t analyzed karanjin yet, but we have had excellent results with isoflavones. Please have a look at the results in this link.
I need information regarding seperation of ions ( Sulfamate and Sulfate) on conductivity. Do you have? Please help.
Hi Mahesh, unfortunately, we do not have a good column for the analysis. Probably the ion-exchange column would work well.
Can we use NaOH in C18 column?
Hi Shinde, Basically Yes. Its pH has to be lower than about 10 in the case of Flex Fire columns. Other types of Develosil columns do not allow such high pH. We would need more details on your sample, please submit a contact form for further details.
nice post! what column best for ibuprofen detection in water matrices ?
Thank you for your comment. Here is a link to a recent app note for pharmaceutical analysis which includes ibuprofen. Please let us know if you have any more questions at firstname.lastname@example.org and a representative will contact you.
Share Information about which molecules can be seprate by which column.
Here is a link to our different applications. You can find which column works best depending on which compound you are interested in. Please let us know if you have any more questions at email@example.com and a representative will contact you.
Which column is best suitable for separating small oligosaccharides and sugar molecules? C18, C8 or C4?
If the compounds are mono or di-saccharides, you can use:
For maltooligosaccharides you can use the following columns in reverse phase. However, anomers will be separated too:
・HSR AQ C18
For related substance of polymyxin B Sulfate which column will be suitable.
We recommend FlexFire WP C8 or C18. The attached file is the example of Colistin which is also a cyclic peptide.
Nicely explained…. I appreciate it… I too have some doubt from the above description, of it is so what is the difference between C18 and H Column or Chiral Colmns…
Which column is recommended for Lactic acid Detection and Estimation?
Chiral columns are used to separate different stereoisomers of the same compound. Unlike the linear alkanes used in C18 columns, the stationary phase of chiral columns is usually one stereoisomer of a polymer such as an oligosaccharide.
For Lactic Acid We recommend FlexFire C30 and FlexFire AQ C18
Thank you for this information. I have been using HPLC for normal phase for a while but presently I need to use a reverse phase for purification of protein. My worry is if I can use the same machine for a reverse phase? If yes, how can I collect a purified sample from the process? The whole thing is quite confusing!
Hi Charles, Before switching between normal phase and reverse phase HPLC, at a minimum the system should be flushed with 100% isopropanol. This must be done because the solvents used for normal phase chromatography are not miscible with the water and buffers used during reversed-phase. If your reverse-phase mobile phase contains any salts or buffers, you should flush again with 100% water before introducing the new mobile phase. These flushes should be done with the column removed. The manufacturer of your HPLC may have further guidance.
Simple, basic good information. Thanks.
I am using Agilent Hi-Plex H, 300 X 7.7 mm column for a mixture of organic acids like Lactic,Acetic, 2-Hydroxybutyric acid, 3-Hydroxybutyric acid,Alcohols like Ethanol, Propanol, Butanol, 2,3-Butandiol, 1,3-Propanediol, Ethyele glycol, MES buffer etc about 31 compounds mix. with about 40minutes runtime with some analytes are almost coeluting.
Any column you would suggest? Any Fused core column can help or any carbon column can help?
unfortunately, our product line does not cover this type of application. We would recommend using ion exchange polymer columns.
The Develosil Team
C18, C8, C4 are universal columns ??
Why can’t we prepare C10 C14 columns ?? Is there any criteria for same ??
Thank you for your question. There is no doubt that C18 and C8 are the mainstream. Additionally, C4 is almost universally accepted as the go to choice for wide pore columns. It is not that we cannot create C10 or C14 columns, there is just little demand for them. We recommend using a C8 column instead of C10 column and C18 instead of C14. Please let us know if you have any further questions. We are always happy to help.