What Is C18? A Guide on the Basics of HPLC Columns

What Is C18? A Guide on the Basics of HPLC Columns

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.

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.

Column Dimensions

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 octadecyl) 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.

The Common Problems of HPLC Columns and How to Deal With It

The Common Problems of HPLC Columns and How to Deal With It

Having troubles on HPLCs can be incredibly frustrating. You’re trying to get some analysis done, and your research can’t move forward until you figure out why your tools aren’t working.

There are some common problems of HPLC columns that pop up from time to time. Knowing what these are and how to fix them can save you hours of frustration. Read on to discover some of these problems and how to address them. We limitted this article in basic level. We would recommend receiving consultation with the manufacturer of your HPLC system.

No Peaks

There can be several factors that cause no peaks or very small peaks to show up on your HPLC outputs. Normal readings should have large, thin peaks that may vary somewhat in height. Small peaks or no peaks at all may mean your detector lamp is turned off, you have no mobile phase flow, your sample is missing or deteriorated, or there’s a problem with your detector, integrator, injector valve or recorder.

Start by making sure your detector is turned on, and then check all the electrical connections and cables. Make sure your autosampler vials have enough liquid and that there are no air bubbles in the sample, and recheck the system with a new standard solution. If that doesn’t work, check the attenuation or gain settings status, and auto-zero if you have to.

No Flow

If you are getting absolutely no peaks on your output, you may have no flow in your HPLC column. This may mean your pump is off or the flow is interrupted or obstructed somehow. You may also have a leak or air trapped in the pump head.

Start the pump if it’s off, and check the mobile phase levels in the reservoir and flow throughout the system. Check the sample loop for any obstructions or air locks, and make sure the mobile phase components are miscible and the mobile phase is properly degassed.

From there, move on to checking the system for loose fitting and the pump for leaks or other issues. Disconnect the tubing at the guard column if you have one, and check for flow. If you’re still having problems, purge the pump at a high flow rate, prime the system, loosen any check valves your system may have, and, if all else fails, flush the system with 100 percent methanol or isopropanol.

Pressure Issues

If your pressure is lower than usual or you have no pressure, you may have a leak or air trapped somewhere in the system. You might also have a faulty check valve or an obstructed or interrupted mobile phase flow. If your system pressure is too high, there’s likely a problem in the pump, injector, in-line filter, or tubing, or you might have obstructed guard or analytical columns.

For low pressure, start by checking the whole system for leaks, loose fittings, faulty pump seals, or bad valves. If everything looks okay, run the same mobile phase check we discussed earlier and check for flow at the analytical and guard columns. If that doesn’t work, reconnect everything and try pumping solvent at double the flow rate.

For high pressure, start by removing the guard and analytical columns from the system and replacing them with unions to reconnect the injector to the detector. Run the pump at 2-5 mL/min, and work on isolating the cause, starting with the detector, then the in-line filter and working back to the pump. If the problem seems to be with the analytical column, reverse and flush the column while it’s disconnected from the detector, and change the inlet frit or replace the column if necessary.

Split Peaks

If you start seeing peaks show up that dip down in the middle, making an M shape, you may have some contamination on your guard or analytical column inlet. You may also have a partially blocked frit or an uneven void at the column inlet. It’s also possible that your sample solvent is incompatible with the mobile phase.

If you think you have contamination at an inlet, reverse and flush the column and change the frit if needed. You can also repack the top of the column with pellicular particles of the same bonded phase functionality and continue using the column in reverse flow direction.

If you think the problem is with the sample, adjust the sample in the mobile phase. Difference of pH in the sample and mobile phase causes split peaks.

Tailing and Fronting (Leading) Peaks

You may notice that rather than coming up and down in straight lines, your peaks start to develop a slight slant at the front or back, called fronting or tailing. This usually means your guard or analytical column may be worn out or your column may be overloaded. Tailing may be caused by a contaminated or deteriorated mobile phase or interfering mobile components in the sample, while fronting may result from problems with the sample solvent.

If you have tailing peaks, start by removing the guard column and attempting analysis, replacing it if necessary. You may also need to restore or replace the analytic column. Be sure to check on the make-up of the mobile phase and the column performance.

If you have fronting peaks, try injecting a smaller volume or diluting the sample. If your solvent is the problem, adjust the solvent, and flush polar bonded phase columns with 50 column volumes of HPLC-grade ethyl acetate at two or three times the standard flow rate, and then with intermediate polarity solvent before you start analysis. You may also want to try another column type.

Negative Peaks

If you see negative peaks start showing up, it’s possible that your recorder leads are reversed. You may also have a refractive index of solute less than that of the mobile phase or a sample solvent and mobile phase that vary greatly in composition. Your mobile phase may also be more absorptive your sample components to UV wavelength.

Check the polarity of your recorder leads to make sure they aren’t reversed, and then try either reversing the leads or using a mobile phase with a lower refractive index. You can adjust or change your sample solvent, and it’s a good idea to dilute your sample in a mobile phase whenever possible. If the problem is with UV wavelength, change the polarity when you’re using indirect UV detection, or use a mobile phase that adsorb your chosen wavelength.

Learn How to Resolve Problems of HPLC Columns

Running down the problems of HPLC columns can be frustrating, but remember, it’s like any other problem you’re trying to solve. Think critically about what might be causing the problem, start eliminating possibilities, and follow the suggested solutions here. You’ll have your HPLC columns back up and running in no time.

If you’d like to find HPLC columns that will stand up to your analysis, check out the rest of our site at Develosil. We have a wide range of HPLC and UHPLC columns with various phase chemistries. Check out our FlexFire series, the column designed for scalability and transferability today.

How to Transfer HPLC Methods to UHPLC

High-performance liquid chromatography is an important analytical tool for a variety of research applications. One of the downfalls of this system is the time it takes to get results. What if we told you there was an HPLC system that generates the accurate results you need without taking all the time?

That’s exactly what UHPLC systems can do for you. But switching to these systems will require some adjustments in your operation. Read on to learn how to switch from HPLC to UHPLC and why you should. More potential problems because of the HPLC column materials are explained in another article. 

What Is UHPLC?

Ultra-high-performance liquid chromatography is a variation on the usual approach to liquid chromatography. They make use of increased operating pressures possible, which yields more accurate results in much less time. Higher pressure makes it possible to use particle sizes smaller than 2 µm, which opens a whole new host of possibilities.

Smaller particle sizes mean you can maximize the number of theoretical plates and make your column lengths shorter. You can also expand your optimum range of mobile phase linear velocities. This means you can use those higher velocities without sacrificing separation quality and get the results you need in much less time.

Column Dimensions

When you’re scaling down in size from an HPLC system to a UHPLC system, you’ll need to adjust the run conditions for the new column. There are some simple calculations you can do to handle this conversion. The first thing you’ll need to calculate is the new column length.

The equation to adjust the column length from HPLC to UHPLC is as follows:

L­C2 = (LC1 x dp2) / dp1

In this equation, LC is the column length, and dp is the particle size.

This equation allows you to maintain the same separation in a shorter column length.

Injection Volume

Now that you have the column length you’ll need, you can adjust the injection volume. When you decrease the column internal diameter and length, you decrease the overall volume and sample capacity, too. You’ll need to be sure to match the sample solvent to the starting mobile phase composition since the column volume is smaller.

To calculate the new injection volume, VI2, you’re going to multiply the original injection volume, V­I1, by the decreased column area. So the equation will read

VI2 = VI1 x [(dC22 x L­­C2) / (DC12 x LC1)]

If you’re using a larger injection volume than you calculated, check for peak abnormalities and irreproducibility that could result from phase overload.

Flow Rate

Flow rate is the volume of mobile phase that travels over time (mL/min) and is the next thing you’ll need to adjust. Linear velocity is the distance that mobile phase will travel over time (cm/min). In order to keep the same linear velocity in a new column, you’ll need to decrease your flow rate proportionally to the decrease in the column internal diameter.

In order to calculate your new flow rate, you’ll multiply your old column flow rate by the square of your column diameter decrease. So your equation is

FC2 = (dC2 / dC1)2 x FC1

in which FC is your column flow rate and dC is your column diameter.

The equation we just discussed is useful for maintaining the same flow rate in a decreased column size, but one of the huge advantages of using UHPLC is they can handle a much higher flow rate. But when you’re working with gradient elution, direct substitution of a higher flow rate isn’t practical. In order to maintain separation under gradient conditions, you have to maintain a comparable relationship among gradient time, mobile phase composition, and column linear velocity and geometry.

Time Program

Now that everything else has been scaled down for the UHPLC requirements, we need to adjust the time program for gradient elution. This will keep the phase interactions the same.

In order to calculate the new time program, you’re going to multiply the differences in the column flow, the column diameter, and the column length by the old gradient time. So the equation is

tg2 = tg1 x (FC1 / FC2) x (dC22 / dC12) x (LC2 / LC1)

in which tg is gradient time, FC is column flow, dC is column diameter, and LC is column length.

You can substitute increased flow rates into the equation to find a comparable time program if you like. But it’s important to keep in mind the system limitations. Mobile phase changes can be limited by mixing chamber capability, dwell volume, and pump accuracy.

Detector Settings

The other thing you’ll need to check when you’re scaling from an HPLC system to a UHPLC system is the detector settings. The data collection rate and time constant must be adapted to the narrower peak shapes. As a general rule, you want each peak to be defined by at least thirty data points.

Depending on the software you use, you may not need to manually adjust the detector settings for the new UHPLC system. Software like the Thermo Scientific Chromeleon Chromatography Data System has a wizard that will take care of calculating the best settings for you. If you don’t have this, check your detector operation manual for further instructions.

Data Collection Rate

As we said earlier, a typical peak requires thirty data points for collection. But when you switch from HPLC to UHPLC, the peak volume and peak width usually decrease. Therefore you must adjust your data collection rate to meet the requisite thirty data points.

To get your new data collection rate, you’re going to divide the old data collection rate, D2, by the square root of the new column length multiplied by the new particle size over the old column length multiplied by the old particle size. The equation will read

D2 = D1 / √ [(L2 x dp2) / L1 x dp1)]

in which D is the data collection rate, L is the column length, and dp is the particle size.

Transfer to UHPLC Today

UHPLC systems can offer some great advantages over traditional HPLC systems. But you will have to do a little footwork to convert your old operations to the new systems. Make sure to account for the capabilities and limitations of your new UHPLC system and you’ll be amazed at the results you can get in a very short time.

If you’d like to find the best UHPLC columns, check out the rest of our site at Develosil. Our columns deliver supreme durability and maximum flexibility. Check out our UPHLC columns today and start getting more accurate results in less time.

A practical example of method transfer from HPLC to UHPLC is explained in another article. 

The Types of (U)HPLC Columns and Applications Sold on the Market Today

The Types of (U)HPLC Columns and Applications Sold on the Market Today

When you’re trying to analyze what’s in a certain mixture, high-performance liquid chromatography is one of the best tools at your disposal. This method runs your mixture through a sort of filter to pull out different molecules that may be in your sample. From there you can look at ratios and determine exactly concentration of your target molecules.

But not all HPLC columns work the same way. Some are based off of molecule polarization, while others have to do with size. Read on to learn about the different HPLC and UHPLC columns on the market and what each is used for.

What Are HPLC and UHPLC Columns?

High-performance liquid chromatography is a method for figuring out which substances are in certain mixtures. HPLC columns act somewhat like a filter to pull out each separate component as it passes through. Different layers in the column have different properties that will attract various substances, so by the time your mixture has flowed all the way through, you’ll know what substances were in it and how much of each there was.

Ultra-high-performance liquid chromatography provides even more resolution in their results than traditional HPLC. They have shorter column lengths and smaller sub-2-micron particles in the columns. They use less solvent and produce less waste, but they are more expensive on the front end.

Ion Exchange

Ion exchange HPLC columns use particles’ ionic charge to separate out the different substances in a mix. The packing in these columns is charged in order to separate out polar molecules. An ion exchange column may be either cationic or anionic, and the molecules are suspended in an aqueous buffer.

Ion exchange HPLC columns are useful in separating out amino acids, carbohydrates, and proteins. Amino acids are zwitterions with both carboxyl and amino groups in their structure, so cation exchange works for them. Carbohydrates can be separated with an anionic column, and proteins will use either cationic or anionic, depending on their net charge.

Ligand Exchange

Ligand exchange HPLC columns target compounds that are able to form labile connections with transition metal cations. The stationary phase is an ion exchanger that carries those transition metal cations. You’ll often see these columns combined with ion exchange HPLC columns; they both contain polarized packing.

This type of HPLC columns can separate monosaccharides, which are smaller units of carbohydrates. They can also separate peptides and amines from amino acids and proteins.

Ligand exchange HPLC columns can separate enantiomers, among other things. This is useful for things such as developing new medications, since you can determine if there will be enough active components to carry out the desired function.

Reversed-Phase

Reversed-phase HPLC columns are the most common in use today. They do not have polarized packing, unlike the ion or ligand exchange columns. The retention times and selectivity of these columns are dependent on a number of other factors, including the pH of the mobile solvent.

The mobile phases for reversed-phase HPLC columns are aqueous and water-miscible organic solvents. The most common solvents you’ll see for these columns are acetonitrile, methanol, and tetrahydrofuran. You’ll see both gradient and isocratic elution used with reversed-phase columns.

Normal-Phase

Normal-phase HPLC columns do have polar packing, unlike their reversed-phase cousins. These columns use a kind of partition chromatography that uses hydrophilic (water-loving) interaction liquid chromatography (HILC). The mobile phases in HILC contain a low amount of water.

The retention time and polarity of analytes in normal-phase HPLC columns can change with the addition of ionic compounds. These columns use organic solvents since normal-phase HPLC columns do not have polarized packing. They are most commonly used for organic acids, biomolecules, drugs, and other small molecules.

Size Exclusion

Size exclusion HPLC works much more like a traditional filter than the other column types we’ve discussed. They filter out different compounds based on molecular size. The packing contains both mesopores and micropores that are distributed in different ways to determine the size of the molecules that can diffuse into the pores.

The retention time and elution profile of size exclusion HPLC columns depend on the number of molecules that can diffuse into those pores. Larger molecules move through the column more quickly, eluting as a single peak after the void volume. So these filters are used primarily for larger molecules such as proteins and carbohydrates.

Application-Specific

In addition to these larger categories of HPLC columns, there are also some application-specific columns that may have customized packing. For instance, aminoglycoside separation can create very harsh conditions that cause columns to break down and need replacing more quickly. Certain columns are designed to withstand these conditions, keeping your columns in good order for longer (which, as anyone with a research budget will be able to attest, is crucial to successful outcomes).

Some application-specific columns can separate pharmaceutical drug substances and their counter ions simultaneously. Others can separate all classes of surfactants out for you. Still others will separate herbicides like paraquat and diquat simultaneously, and some can perform rapid analysis of polyaromatic hydrocarbons. 

Find the Right HPLC Columns for You 

Every type of HPLC column functions differently and has variable applications. Knowing which HPLC columns are on the market is crucial when designing you analysis and generate more accurate results.

If you’d like to get the best HPLC columns on the market, check out the rest of our site at Develosil. Our FlexFire Series delivers supreme durability and maximum flexibility. Learn more about this series today and discover the transferability these columns can offer you.