The dissimilarities between HPLC and GC were discussed in the earlier article- How are Gas chromatography (GC) and High Performance liquid chromatography (HPLC) different?. The present article discusses 10 key features which are common to both the techniques. On going through these features you will recollect your basic understanding of chromatography and appreciate the commonality between the two most popular separation techniques.
1. Common basis
Separation of components of a mixture, their identification and quantification is the common underlying objective of both the techniques. This is achieved through a choice of columns, mobile phases, stationary phases, detectors and operating conditions.
2. Role of Mobile Phase
The mobile phase plays an important role in transporting the sample to the separating column and its separated components to the detector in a sequential manner. While both HPLC and GC function on the same principle the only difference is that HPLC uses liquid mobile phase whereas GC employs a gas as the carrier.
3. Sample Introduction
Both GC and HPLC are capable of analysis of liquids and dissolved solid mixtures though gaseous mixtures are also routinely analyzed using GC. Sample introduction techniques are common generally common.A gas tight syringe is sufficient for introduction of liquid samples. For gases Teddler bags or auto sampler (manual or automated operation) are used.In HPLC systems the mobile phase is moving under higher pressure so direct syringe injection is not possible. A syringe is used to fill a fixed volume loop in the manual sample introduction valve and with the change of the valve position the entire sample inside the loop is carried by the mobile phase into the column. Auto samplers are a great help for unattended operation when the number of samples is large.
4. Stationary Phases
Separation of the sample components inside the columns takes place on the basis of distribution of sample components between the stationary phase and the moving carrier mobile phase. The distribution is influenced by physico – chemical interactions between the sample components and the stationary phase materials. An exception is Gel Permeation chromatography where separation takes place due to difference in size of the eluting molecules.
5. Band Broadening
Band broadening is a major contribution to loss of resolution as it leads to overlap and merger of otherwise well separated peaks. Van Deemter equation describes the overall efficiency of a GC or HPLC column and takes into consideration factors like diffusion, mass transfer and velocity of mobile phase. The relationship is helpful for understanding of band broadening and optimization of operating conditions for both GC and HPLC separations.
6. Peak Response
Both GC and HPLC chromatograms look similar. The response is peak shaped in both cases and the concentration of the eluting compound is proportional to the peak height or peak area.
Principally four methods are used for quantitative estimations both in HPLC and GC:
- Normalization of Peak areas
- Internal standard method
- External standard Method
- Standard additions method
7. Reference standards
The basis of quantitative analysis is comparison of the sample response with that of a pure reference compound. Such reference working standards need to be validated from time to time by comparison of their specified parameters against standards traceable to certified materials available from internationally recognized standardization bodies. In routine analysis you need to verify the retention time of injected standards on GC or HPLC systems under specified operating conditions.
8. Common Application Softwares
Mostly manufacturers supply common HPLC and GC application software as peak analysis and data treatment are common . This is a big benefit if the systems are procured from the same manufacturer and multi user licences are obtained. Such softwares also help set up operational parameters and method sequencing.
Examples of software provided by reputed suppliers are:
- Perkin Elmer- Total Chrom
- Agilent- Chemstation, EZ Chrom Elite
- Thermo Scientific- Chromleon 7.1
9. Hyphenation with other techniques
Both GC and HPLC have been successfully hyphenated with Mass Spectroscopy to achieve excellent resolutions and high sensitivities. Both the techniques have also been successfully coupled with FT-IR to arrive at structural confirmations on separated mixture components.
10. Expression of Column Efficiency
The efficiency of both HPLC and GC columns is expressed in terms of Height equivalent to theoretical plate(HETP). This value is used in deciding the useful life of both GC and HPLC columns over their usage.
Both GC and HPLC are the pillar stones of chromatographic analytical laboratories and while it is important to know the differences between them it is equally important to understand the similarities so as to decide on the right method for a particular application in hand.