Hyphenated techniques involve physical coupling of instruments through a compatible interface. Hyphenation results in a marked improvement of analysis in terms of speed, specificity and sensitivity. The benefits of coupling are realized mainly in chromatographic separations involving the popular GC and HPLC techniques. Such combinations have received a great deal of attention from analytical chemists for identification and quantification of complex mixtures. The key application areas are:
- Analysis of drugs
- Poisons and their metabolites
- Clinical screening
- Pesticide residue analysis
- Forensic investigations
- Foods and flavours
- Petroleum products
Mass spectral libraries are available commercially for common constituents of several synthetic as well as natural compounds. Such libraries and in-house developed libraries offer positive identification of sample constituents in different product categories.
Hyphenation with Gas Chromatography (GC- MS)
GC – MS combines the separation capability of the gas chromatograph with the resolving power of a mass spectrometer. It was the first hyphenated techniques which helped pioneer separation and quantification of complex mixtures comprising of volatile and semi-volatile compounds which retain their identity at high temperatures. Both GC and MS complement each other but due to pressure range differentials (GC at atmospheric or higher pressures) and MS (vacuum of the order of torr) a suitable interface is needed for transfer of sample between the two instruments without liquid carryover.
CC – MS provides useful information on volatile mixture components, solvent residues, drugs and poisons and the analyst is able to save valuable time with the help of mass spectral libraries for comparing the data from unknown compounds.
Liquid chromatography-Mass spectrometry (LC – MS)
LC – MS is today is an accepted powerful technique which permits identification and quantification to a very high degree of accuracy. The liquid chromatograph with its wide option of separation column sizes, stationary phases and detectors helps separate sample constituents, impurities and other degradation products while the mass spectrometer helps resolve and identify the various molecular mass fragments on the basis of their mass to charge ratios.
LC – MS poses a unique challenge as the mobile phase flow rate is generally around 1ml/min. Such flow rates introduce significant quantity of liquid solvent into the vacuum system which needs to be removed to prevent damage. Different approaches are in use to effectively ionize the molecules and remove the solvent. Electro spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are two main interfaces which are used for majority of applications.
The soft ionization techniques generally used for large molecules such as poly aromatics, peptides and proteins mainly result in the molecular ion and only a small number of fragmented molecules. Significant improvements in number of ionized species have been achieved through collision – induced subsequent dissociation of the resulting molecular ions. Such high degree of fragmentation is achieved through tandem mass spectrometry (LC – MS- MS). This technique is gaining popularity due to its capacity to provide high levels of separations and low detection limits. Its key features and benefits will be covered in subsequent articles.