Critical points to be considered during Method development by Gas Chromatography

Gas Chromatography (GC) is a technique used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Gas Chromatography is widely used in analyzing agro-products, petrochemicals, chemicals, pesticides, and pharmaceuticals. The selection of stationary phase, carrier gas, column oven temperature, injection technique, handling of the sample matrix, and sample diluent are a few critical components of the method development process. In this article, we will discuss three critical components: the injection technique (Liquid Injection Vs Head Space), handling of the sample matrix, and sample diluent.

1)???????Liquid vs Head Space:

What is the liquid injection and head space?

Liquid injection: The sample solution gets injected in a liquid form inside the injector port with the help of a syringe. The liquid gets vaporized inside the injector port due to sufficiently high temperature.

Headspace: In the Headspace technique, only the gas phase above the sample is introduced into the GC column through the transfer line. The headspace technique is based on Henry's law, which states that the ratio of a dissolved substance in a solution to the substance in the vapor in a sealed glass vial is constant, depending on temperature.

What are the advantages of liquid injection and headspace?

Liquid Injection: The liquid injection can result in a shorter analytical time as compared to the headspace. The cost of analysis with the liquid injection technique is low compared to head space.

Headspace: Headspace can minimize column contamination. Often higher sensitivity with good precision and linearity. The sample matrix interference can be minimized to the great extent in HS. The sample in its solid state also can be analyzed by using HS. Moreover, the undissolved analyte can be well extracted by using HS.

What are the disadvantages of liquid injection and headspace?

Liquid injection: In liquid injection, the column can get irreversible damage due to non-volatile compounds present in the sample. The injection repeatability can be a challenge for highly viscous samples.

Headspace: The analysis time is quite high as the sample needs to be heated for a certain time (such as 30 minutes). A system with accurate pneumatic control is a need of the HS.

How to select the right injection technique?

In case you are handling a drug product containing a huge sample matrix, headspace can be the preferred injection technique. However, in case the sample is clean and there is no decomposable sample matrix, liquid injection can be preferred.

2)??Sample matrix minimization:

The sample matrix of the pharmaceutical drug product may pose a great challenge to the analytical column while GC analysis. Similarly, the extraction of the analyte can be at the stake due to a dirty sample matrix. The analyst has to tackle these two critical challenges during method development. The HS can be of great help in eliminating or minimizing dirty sample matrix as well as extraction efficiency. The thermal treatment can help in increasing extraction efficiency and the design of HS will help in avoiding sample matrix. The sample matrix can also be managed by using precipitation, filtration, and centrifugation.

3)??Diluent selection:

In GC, the diluent can impact the reproducibility and response of the analyte. The diluent can also result in the Backflash, which may contaminate the injector and result in a ghost peak or distributed baseline. The solvents with high molecular weight (such as DMSO, and Benzyl alcohol), resulting in low gas volume and hence may not result in backflash in comparison to low molecular weight solvents (such as water, and methanol). The viscosity of the diluent can be critical in the case of liquid injection as repeatability (due to loss of precise injection volume) may get compromised. Adequate precautions must be taken while handling high viscous diluent while using the liquid injection technique. The “focusing of the analyte” is also dependent on the diluent selected. The disturbed peak shape of the early eluting compound sometimes results from poor column focussing. The response of the non-polar analyte can get drastically varied from one injection to other if water is used as a diluent.

What is backflash and its impact on GC analysis?

Backflash happens in the GC injection port when the sample’s solvent expands as it’s vaporized to a volume that is larger than the injection port volume. When this happens, the sample flashes back up to the top of the injection port and can cause a multitude of chromatographic problems such as leads to poor reproducibility, sample loss, ghost peaks, carry-over, split peaks, tailing peaks, loss of resolution.

How to prevent backflash?

To help prevent backflash, consider the below ideas

??Use a sample solvent with a higher molecular weight.

??Use the minimum sample injection volume which to reach the required detection and quantitation limits.

??Use an injection port liner with a larger volume.

??Use the lowest reasonable injection port temperature.

What is Solvent Focusing?

The purpose of solvent focusing is to split the injection to get a sharp peak because it allows for greater sensitivity without sacrificing resolution. Solvent focusing can be used to obtain sharp and symmetrical peaks for the majority of analyses by correctly setting the GC parameters.

Solvent focusing is an effective method for obtaining sharp peaks. It is of particular value when low detection limits are required. Three parameters can effectively improve the technique:

??the initial column temperature,

??the column phase ratio, and

??the boiling points of the sample components.

?From the above three, changing the initial column temperature is the quickest way to effectively attain solvent focusing and get narrow or sharp peaks and lower detection limits.