Many chemical compounds, including drugs and metabolites, can be analyzed by either gas chromatography (GC) or high-performance liquid chromatography (HPLC). Because both techniques function under the same basic principles of compound separation, identification, and quantification, it can sometimes be tricky to choose one over the other. But knowing what differentiates HPLC and GC can definitely help with the process.

So let me highlight some of those differences for you:

Nature of Samples

GC responds to substances that can experience a change in matter to the gas phase with heat – that is, volatile compounds. Therefore, GC is limited to compounds that are low in molecular weight and stable at high temperatures. So, if you’re working with proteins, forget it, GC is not for you. An alternative to non-volatile small molecules, such as amino acids and sugars, is to make them volatile by chemical derivatization1.

Now, your other option is HPLC. You can analyze volatile and non-volatile compounds, from low to high molecular weights, as long as they are soluble in a liquid phase. Also, you don’t need to worry about thermal stability since samples usually run at room temperature.

Surely, the wide sample scope offered by HPLC is one of its most attractive features. But if both techniques are still feasible for your sample, don’t make a decision yet – there’s more to consider.

Mobile Phase

As implied by their names, the mobile phase for HPLC is liquid and for GC is gas. Another major difference is that the mobile phase is a key participant in sample separation for HPLC but not for GC.

HPLC uses solvent mixtures and gradients. The polarity, solubility, and complexity of the sample are some of the factors that influence the selection of the mobile phase. To be clear, choosing the mobile phase is a crucial part of HPLC and troubleshooting for better separation is often complex.

In contrast, GC uses a pure inert gas as the mobile phase. It’s commonly known as the carrier gas because its purpose is to carry molecules through the column. Typically, the carrier gas used is chosen based on the detector, so it’s not something you have to worry about.

Resolution

If your sample is a mixture, bear in mind that resolution – a measurement of how well two peaks are separated – is affected by a number of factors. But it comes down to how similar the partitioning equilibrium between the mobile phase and the stationary phase is for two compounds. The more comparable the partitioning equilibrium is, the higher the chances of having co-elution. If this happens, you may not be able to see where one peak ends and the other starts. So the resolution would be poor.

Since GC separation is based on compound volatility, similar molecular weights can result in comparable retention times and cause the peaks to overlap. In the same way, the polarity of molecules affects the resolution in HPLC – that, and also the composition of the mobile phase.

Columns

HPLC columns are short, wide, and made with tightly packed material. In contrast, GC columns are long and narrow, and come in two general types; namely, packed columns and capillary columns.

Capillary columns have many advantages over packed columns, including improved resolution and speed. Because of this, capillary GC is usually preferred over HPLC (when possible) for the analysis of highly complex samples.

Detection

Detectors respond to a molecule’s chemical or physical properties. Some detectors are universal – they can detect all (or most) compounds – and others are selective by responding to a specific property. So, detection can limit your options depending on which detector is available to you, and whether your sample has the necessary characteristics for its detection.

GC detectors vary in selectivity and sensitivity2. The flame ionization detector (FID) and the thermal conductivity detector (TCD) are two common examples. The first is selective to hydrocarbons, whereas the second is universal.

If your lab has an HPLC, chances are that it has an ultraviolet-visible (UV/Vis) spectroscopic detector3, which responds to light-absorbing molecules such as aromatics. The refractive index detector (RID) is also commonly used because it has universal detection capabilities. Yet it suffers from low sensitivity and incompatibility to gradient runs, so it isn’t ideal for complex samples.

Undoubtedly, the most powerful detection method is that offered by GC/MS and HPLC/MS (or LC/MS). These analytical systems combine the features of the chromatograph with that of a mass spectrometric (MS) detector. So, one part separates components and the other provides mass analysis for each of those components – giving you additional information for compound identification.

Relative Cost

If money is an issue, you may want to consider that a tank of gas is generally cheaper than solvents. In addition, because liquids are more viscous than gas, HPLC relies on pressure pumps to force the mobile phase through the column. Since a pressurized system is more complex, this adds to the instrument’s costs of operation. GC on the other hand is not very complex. Simply think of it as a large, sophisticated oven that requires a lower degree of maintenance.

The table below summarizes the differences described above and lists typical sample types for both chromatography techniques.

Comparison Between GC and HPLC

 GCHPLC
SampleVolatile
Low molecular weight
Stable at high temperatures
Volatile/non-volatile
Low/high molecular weight
Stable at room temperature
Soluble in liquid phase
Resolution issuesSimilar molecular weightsSimilar polarities
ColumnLong and narrow
Packed column
Capillary column
Short and wide
Packed column
Mobile phasePure inert gas
No gradient
No pressure
Solvent mixture
Gradient
Pressure required
Optional detectorsFID, hydrocarbons
TCD, universal
GC/MS, mass
UV/Vis, light absorption
RID, universal
LC/MS, mass
Relative costLowHigh
ApplicationsOils, plant pigments, pesticides, fatty acids, toxins, air samples, drug abuse testing (i.e. cocaine)Inorganic ions, polymers, sugars, nucleotides, vitamins, peptides, proteins, lipids, tetracyclines

Always keep in mind that both HPLC and GC are invaluable advancements in analytical instrumentation and indispensable in many scientific fields. But they are also susceptible to issues and limitations. Common problems are related to selectivity, sensitivity, and resolution. And these are affected by the nature of the sample.

So when deciding between the two, it’s really not a question of instrument superiority, but of effectiveness to the sample of interest.

My advice to you is to evaluate your sample based on the factors described in this article and weigh your options with as much information as you can get. And now it’s up to you to make the final decision!

Are you going to run your sample on HPLC or fly it through GC?

References

  1. Ahuja S (1976) Derivatization in gas chromatography. J Pharm Sci. 65:163–182.
  2. Hartmann CH (1971) Gas chromatography detectors. Anal Chem. 43:113A–125A.
  3. LaCourse WR (2002) Column liquid chromatography: equipment and instrumentation. Anal Chem. 74:2813–2832.