Capillary liquid chromatography

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Capillary liquid chromatography is a new edition in the accelerating advancements witnessed in chromatography over the last decade. It offers a diverse array of powerful applications for an analytical chemist. Having said that, small-scale, highly sensitive bioanalytical separations are a specialty of capillary liquid chromatography.

What is capillary liquid chromatography

Liquid chromatography performed in specially designed capillaries as opposed to the conventional glass or stainless-steel analytical columns is called capillary liquid chromatography. It is a micro-column technology. Capillary columns have small dimensions such as a smaller inner diameter, below 500 µm. A specific sample volume is injected into the capillary columns.

The small internal diameter of the capillary enables an accurate mobile phase flow rate in µL/min or nL/min. Extremely well-resolved chromatographic peaks are obtained as the end result in capillary liquid chromatography hence ensuring a precise, desirable sample separation.

Different types of capillary columns

Capillary columns generally fall under two different categories.

  1. Open tubular capillary column: The capillary columns in which the stationary phase is coated along the inner walls. The center of the column is hollow/open allowing an unrestricted path for the mobile phase solvent to flow.

The open tubular capillary columns are further classified into sub-types depending upon the different types of stationary phase coatings.

  • Porous layer open tubular (PLOT): A porous layer of solid stationary phase particles is coated along the inner walls of the column. The porous stationary phase particles may be organic, or inorganic in nature. Porous polymers and molecular sieves are often used which provide resistance to harsh mobile phase conditions.
  • Wall coated open tubular (WCOT): A liquid stationary phase is coated along the inner walls of the column without any surface support.
  • Surface/support coated open tubular (SCOT): The inner wall of the column is coated with a liquid stationary phase using a solid support.

2. Fused silica capillary column: It is the most frequently used capillary column. A polymeric stationary phase such as polyimide is bonded to the outer column wall. This provides high strength, durability, and great flexibility to the column. The inner wall of this column is coated with a liquid stationary phase and fused silica gel.

What is the stationary phase in capillary liquid chromatography

Silica is the first choice as a stationary phase material in capillary liquid chromatography like all other types of liquid chromatography. Other than that, polymeric substances such as squalene (C5H8)6, polydimethylsiloxane (C2H6OSi)n, and Apezion L. can also be used as stationary phase coatings for open tubular capillary columns.

The stationary phase can be physically adsorbed onto the capillary column. In this case, the column wall acts as an adsorbent while the stationary phase is the adsorbate. Physical interactions depend upon the capillary surface and may include electrostatic forces or hydrophobic forces of interaction.

In fused silica capillary columns, fused silica acts as the adsorbent on which the liquid stationary phase molecules get adsorbed. The stationary phase can be adsorbed as a  monolayer or in a layer-by-layer arrangement. 

What is the mobile phase in capillary liquid chromatography

As its name implies, a liquid mobile phase is used in capillary liquid chromatography. Combinations of organic and aqueous solvents are used as mobile phases. This may include acetonitrile-water or a methanol-in-water combination. Both an isocratic as well as a gradient mode of elution can be used in capillary liquid chromatography.

How to perform capillary liquid chromatography

Instead of performing high-performance liquid chromatography (HPLC) with the traditional stainless-steel column packed with silica gel, a capillary column can be used and the HPLC method modified for the analysis and separation of small sample volumes. All the other components stay the same.

Here is a step-by-step guide on how a capillary liquid chromatographic analysis is performed:

Step I: Column packing

A high-pressure and/or sonication is often required to ensure efficient column packing in capillary columns. Small dimension (100-350 µm x 25 cm) columns are used. These capillary columns are housed in cartridges which are then incorporated into the chromatographic set-up.

Step II: Sample injection

About 60 nl sample size is injected into the analytical column with the help of an automatic injector system.

Step III: Mobile phase flow

High-pressure syringe pumps are used to facilitate a capillary-scale mobile phase flow through the column. The flow rate is maintained at approx. 20 µL/min while an ultra-high pressure up to 10,000 Psi can be applied. As the system is based on a capillary column so there is a reduced chance of developing back-pressure inside the column, unlike the conventionally used packed columns.

Step IV: Analyte separation

Analyte separation occurs inside the column in a normal or a reverse-phase mode. Components with a high affinity (strength of adhesion) with the stationary phase coating get retained while those with high solubility in the mobile phase are eluted out of the column.

Step V: Detection

A mass spectrometry (MS) detector is coupled with the capillary column for component detection and identification. The detectors receive sample components, readily detect them, and send a signal to the recorder. The recorder then plots a chromatogram.

A chromatogram with well-resolved peaks is obtained in capillary liquid chromatography.

Major differences between capillary liquid chromatography and other liquid chromatographies

FeatureConventional liquid chromatographyCapillary liquid chromatography
Column typePacked stainless steel
or glass analytical scale column
Open tubular
or fused silica-coated capillary column
Column diameter1 to 4.6 mmUnder 500 µm
Mobile phase
 flow rate
High
 (0.2 to 2.0 mL/min)
Significantly lower
 (2-20 µL/min or in nL/min)
Sample loading capacityHighLow
SensitivityLow sensitivity for trace componentsHigh sensitivity for trace components
present in a sample

Applications of capillary liquid chromatography

  • Capillary liquid chromatography-mass spectrometry allows bioanalytical separations using cells, tissues, and biological fluids as sample matrices.
  • Capillary liquid chromatography finds meaningful applications in clinical research such as proteomics i.e., it can isolate and identify proteins present in trace quantities in a biological fluid used as a sample. Genetically modified proteins can be analyzed with high resolution and sensitivity using capillary liquid chromatography.
  • It can also be used in drug analysis, studying food contaminants and environmental pollutants present in small quantities, otherwise difficult to separate via traditional liquid chromatographic techniques like HPLC.
  • Compact capillary columns can also be used in small-scale forensic analysis.

Limitations of capillary liquid chromatography

  • Capillary columns offer a low sample loading capacity at a time.
  • Capillary liquid chromatography is relatively a newer technique. There are limited detector options available that are compatible with this technique. Due to low sample load-ability, detectors with high sensitivity i.e., a low LOD value are required. The MS detector is the most feasible choice so far.

Conclusion

In conclusion, capillary liquid chromatography has a long way to go. It offers higher sensitivity (about 2000 times higher) than the conventional liquid chromatographic techniques for the separation and analysis of trace quantities of analytical components.

You can also have a look at our article in which we talked about the capillary and open tubular columns for gas chromatography.

For more extensive and specific information on capillary liquid columns, consult this:  capillary columns in liquid chromatography.

References

1. P.Grinias, J., J. M.Godinho, F. Gritti, K. A.Schug, D. S.Bell and P. K.Dasgupta (2020). State-of-the-Art in Capillary Liquid Chromatography (LC): Now, Next, and How? LCGC North America 38.

2. Wilson, S. R., H. E. Berg, H. Roberg-Larsen and E. Lundanes (2020). Chapter 3.3 – Hyphenations of one-dimensional capillary liquid chromatography with mass spectrometry: State-of-the-art applications. Hyphenations of Capillary Chromatography with Mass Spectrometry. P. Q. Tranchida and L. Mondello, Elsevier: 319-367.

Capillary liquid chromatography

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