Chromatography - International Union of Pure and Applied Chemistry has defined the chromatography as 'it is a technique which is primarily used for the separation of the components in the sample, where the components of the sample are distributed between two phases, which are the stationary and mobile phases. Solid, gel and liquid supported on a solid material can be used as a stationary phase, which may be distributed as a film, spread as a layer or packed in specific columns. Liquid or gas can be used for mobile phase.
High performance liquid chromatography (HPLC) and Gas Chromatography techniques are widely using worldwide for the separation, qualitative and quantitative analysis.
Conventional HPLC is used for identification and quantification of analytes in mixture. Stationary phase can provide much surface area. There are two types of variants can be used in the HPLC depending on the relevant polarity of the solvent (mobile phase) and stationary phase. Silica fused (C18) columns are widely used as stationary phase. Mobile phase is the mixture of solvent (most common are methanol, acetonitrile and phosphate buffer).
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Normal phase HPLC - stationary phase is more polar than mobile phase.
Reversed phase HPLC - mobile phase is more polar than stationary phase.
Conventional Gas Chromatography is a type of chromatography, in which the mobile phase is a carrier gas. Inert gas (helium), an un-reactive gas (nitrogen) or argon is used as a mobile phase. Microscopic layer of liquid or polymer on an inert solid support is used as a stationary phase, which is called as column. GC is used for separating chemicals in a complex sample. Chemical components in the sample pass in a gas stream in different rates because of the various physical and chemical properties of the sample and interaction with stationary phase. This technique is widely used for the separation of volatile compounds.
Highly complex mixture cannot be analysed using conventional chromatographic techniques, especially mixture of peptides and biological fluids. This problem can be solved using multidimensional chromatographic techniques. This technique used multiple columns. Each column contains different stationary phases in it. These columns are coupled together, basically orthogonally. The main principle behind the multidimensional chromatography is one of the fractions (fraction of interest) or fractions from in the first column can be selectively transferred to the second column, where the additional separation takes place and give better separation than conventional chromatographic techniques. These two columns separate the analyte of interested on the basis of different mechanism, for eg. First column separates the on the basis of molecular weight of the molecule and the second columns on the basis on the polarity of the analyte.
Columns temperature might be different. It gives the higher resolution, selectivity and increase in peak capacity, when compare with the traditional chromatographic techniques. And this makes us possible to the combination of LCXLC, GCXGC and GCXLC.
Using multidimensional chromatography, it is possible to characterise a mixture of thousands of components in a single run. Two dimensional chromatography is the most popular version of the multidimensional chromatographic techniques. This is also called as sequential analysis, boxcar chromatography, column switching, coupled column chromatography and multiphase chromatography .The output of multidimensional chromatography is called as multidimensional chromatogram.
There are two modes of multidimensional chromatographic process according to the amount of elute entering from the first column to the second column -
Ideal mode, where all the components of the eluents from the first column enter into the second column and further separation occurs.
Comprehensive mode, where at regular intervals only a small fraction of the eluent from the first column enters into the second column in the form of narrow chemical pulses (focusing step). This transfer interval time can be determined based on the width of the peak, which are eluting from the previous separation dimension. GCXGC is the best example for the comprehensive type of mode.
Multidimensional liquid chromatography:
Two dimensional: As this multidimensional liquid chromatography has two separate columns for the better separation, it needs column switching valves to couple two or more separator columns. These valves are one of the main components in Multidimensional LC. Basically two similar columns are coupled together in series; it makes the 2D-LC distinct from the single dimensional chromatography. But the columns with different pore sizes are coupled together in 2D-gel permeation chromatography to maximize the separation of wide molecular weight compounds in a sample. Simultaneous separation of cations and anions can be done by coupling cation-exchange and anion exchange columns together.
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Three dimensional: In some cases two dimensional chromatography is not enough to separate highly complex peptides in proteome samples. Ultra high peak capacity and ultra peak resolutions are required for the analysis low abundance proteins, which can be achieved by three dimensional chromatography. Eg- Size Exclusion Chromatography (SEC) - Strong Cation Exchange Chromatography (SCX) - Reverse Phase Liquid Chromatography (RPLC).
Column switching: The function of the column switching is to diversion of various sample components to different columns that too from the single injection to achieve maximum resolution in shorter period. It can be generally performed using 6 ports valve by rotating it at appropriate time. It is called as heart-cut techniques. Some times 8 and 10 ports valves are also can be used. When the valve is at one position sample passes through the first column and then to the detector. But when the valve is at another position sample passes through the first column to the second column and then to the detector. When the samples contain the both weakly and strongly retained solutes, then this approach is very help full by choosing column A to have significantly less retention capacity than the column B.
Schematic picture of LCXLC
Columns - columns are made of stainless steel or glass-lined steel tubes.
Stationary phase - silica, chemically modified silica, styrene-divinylbenzene, other metal oxides, agarose and porous graphitic carbon are can be used as the stationary phase. These can be chosen according the requirements of analysis.
Detection in multidimensional chromatography is similar to that in one-dimensional chromatography. Choice of the detector depends on the several things such as, separation mode using for the second dimension, nature of the molecules and the problem is being solved.
UV - UV detection is used for the detection of peptides, chromophores and proteins.
Refractive index detection - is used for the detection of polymers.
Evaporative light scattering detection is used for surfactants, which do not contain UV chromophores and polymers.
Fluorescence Detectors - used to detect the compounds, whatever exhibit fluorescence.
Electrical conductivity detectors - these detectors measures the conductivity of the mobile phase.
Mass spectroscopy - for the high resolution analysis.
Multidimensional Gas Chromatography (GCXGC):
Multidimensional GC is more popular than the Multidimensional LC for the multidimensional separations. In multidimensional GC, columns are connected or coupled to each other in series or parallel. A range of analysis can be done. Selected gas chromatographic fraction is transferred from one column to another column, which is capable of independent control, cryogenic trapping may used. As secondary column is more polar than the first column, substances which have different polarities but same boiling points can be resolved out in the second column. Taps or values used as switching devices between two columns. There are two column switching techniques heart cutting and backflushing. In heart cutting technique, a small fraction of carrier gas enters into the second column, which is similar to multidimensional LC. In GC second column polarity may vary. In backflushing technique carrier gas flow in the first column is reversed, once the interest of component elutes and enters into the second column. This is used for rapidly eluting volatile components of interest in the sample. Low volatile components are flushed back in the first column as they travelled a short way down the column. Using backflushing technique we can increase the life time of the second column, second detectors and can shorten the time of the analysis. Cryogenic focusing and adjusting the relative pressures also have great influence on the entire process.
Packed columns - these columns are made of stainless steel or Pyrex glass coil. These columns give greater precision and accuracy.
Open Tubular columns - these columns are mostly used for the general use. Internal wall of the column is coated with the stationary phase as a thin film 0.2mm to 1 mm think.
Absorbents - silica, Alumina
A wide range of detectors available for multidimensional GC. Selection of the detector is depends on the nature and LOD of the target compound, application in question, matrix, budget, scanning speed of the instrument and cycle time of the analysis. Some of the widely using detectors are
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MS - quadrupole mass spectrometer gives superior spectral integrity and sensitivity
TOF-MS - for the fast detections
Electron capture detector (ECD) - It detects electron absorbing components. Eg- halogens.
Flame ionization detector (FID) - it has a wide range of application. It is widely using for the analysis of petroleum products and hydrocarbons.
Nitrogen/Phosphorus detector (NPD) - These detectors have a long life time and high operational stability. It is widely being used in environmental and forensic applications.
Sulphur and Nitrogen chemiluminescence (SCD & NCD) - widely being used in petro-chemical applications of accurate quantitation of nitrogen and sulphur in fuels.
P-pressure controller; R-restrictor or needle valve; NO and NC-normally open and normally closed position
Schematic picture of GCXGC
Sample preparation of multidimensional GC - All samples must be dissolved in the representative mobile phase (methanol). Lab grade solutions must be used. Sample preparation is depends on the chemical and physical properties of the sample to be analyses. Derivatization must be carried out, when compound are not suitable for direct gas chromatographic analysis as they contain protic hydrogen, phenol, amines etc. The aim to carry out derivatization is to substitution of active functional groups to decrease or increase volatility, thermal stability and polarity of molecules and improves detectability. It can be achieve by 3 methods are;
Acylation - for compounds contain NH, SH and OH groups. Perfluoro acid anhydries, pentafluropropionic anhydride, N-methylbis are some of the reagents are used for acylation.
Alkylation - for modify acidic hydrogens like carboxylic acids and phenols. Reagents are used for alkylation are dimethylformamide and perfluoroalkyl reagents.
Silylation - silylation is substituting of an active hydrogen for a trimethylsilyl-N group. Dimethyldichlorosilane, trimethylchlorosilane and N-dimethylcarbamate are some generan silylation reagents.
Sample preparation of multidimensional LC - Sample preparation is depends on the chemical and physical properties of the sample to be analyses. All HPLC grade solutions must be used. Mobile phase solvents must be degassed before starting analysis to avoid bubble formation in the column. The sample of our interest must be fully dissolved in the mobile phase solution. Any undissolved particles must be filtered out.
With parallel cryogenic traps - parallel cryogenic traps are attached between two serially coupled columns. Heat cutting technique by using parallel cryogenic traps limits the number of cuts to one for a given injection.
MDGC/FT-IR/MS - Coupling with infrared and mass spectrometry. It is very effective for detailed analysis of small subsets of a mixtures (essential oils).
TAG with 2D-GC - Thermal desorption aerosol GC is coupled with 2D-GC to improve the separation of organic compounds present in atmospheric aerosols, such as alkanes, aldehydes, polycyclic aromatic hydrocarbons.
Capillary flow technology - multidimensional GC with the capillary flow technology with a pressurized liquid injection system and highly selective capillary columns is used for the trace analysis of oxygenated compounds.
IEX-RPLC - Reversed phase liquid chromatography coupled with ion-exchange chromatography, is an example for orthogonal two-dimensional analysis. This technique features high sensitivity for the identification of peptides by increased resolution and decrease in overlapping of peptides. The total peak capacity is greater than 5000. IEX provides high sample capacity, while RPLC is compatible with Mass Spectroscopy.
SCX-RPLC-MS/MS - Strong Cation Exchange chromatographic column is coupled with the reversed phase liquid chromatographic column. This technique is widely using for the analysis and identification of proteins. In the first step digested proteins can be separated using SCX column and these fraction are collected for every minute. In the second step fractions collected from the SCX column will be introduced in to the RPLC column, which is coupled with tandem mass spectroscopy.
One of the most recent advances in multidimensional chromatography is Bi-phase columns. Two types of the column materials are sequentially packed together in a single column, SCX and RPLC.
SEC-RPLC - size exclusion chromatographic column is coupled with the reversed phase liquid chromatographic column. This techniques is highly sensitive for the analysis of complex proteins, analysis of proteomics and immunoglobulin fusion proteins.
SEC-SCX-RPLC: This three dimensional chromatography is used when two dimensional approaches cannot give satisfactory results. SEX, SCX and RPLC columns are coupled together for the ultra peak resolution and capacity, which we need to analyse low abundance proteins and proteins with extreme molecular weights.
With monolithic WAX and RP columns: Weak ion exchange monolithic columns are coupled with Reversed phase monolithic columns in micro format in order to analysis biopolymers. These columns offer a lot of advantages like, high resolution, fast mass transfer and low back pressure in very short time frame.
LC2-SPE-NMR - two dimensional liquid chromatography, Solid phase extraction and nuclear magnetic resonance are combined together. Chromatography is for the separation of the compound of interest, SPE for isolation, enriching and to deliver the compound to NMR probe and NMR for analysis.
2D-LC with an innovative solvent evaporation interface - this interface reduces the bandwidth of the primary fraction injected on the second column.
For the both Multidimensional LC and GC, calibration curve is to be plotted concentration against peak area. Need to interpret the unknown value using the equation Y=MX+C, getting on the Ex-cel.
Applications of 2-D-GC - there are several applications in analysis in/of
Food and environmental samples
Pesticides in vegetables, fruits and in environmental samples.
Fat and oils
Amino acids in beer, honey and wine.
Volatiles in roasted coffee beans
Soil, water and sediment
Pesticides, drugs, steroids in blood and serum
Drugs of abuse
Applications of 2-D-LC - there are several applications in analysis of/in
Fat soluble vitamins.
Drug isolation from urine and plasma
Homopolymers, oligomers, copolymers
Polycyclic aromatic hydrocarbons.
Advantages of multidimensional chromatography -
Highly selective and accurate
High resolution and precision
Compatible for a wide range of analysis
Fully automated and sophisticated
Gives very complex data
Requires highly skilled professionals to work on
This technique has been using for the past several years to resolve the selective components from the samples, which are poorly resolved. Multidimensional chromatography features low sample volumes, high pressure multiport switching valves. It has more widespread applications. Analysis of hydrocarbons in petrol samples, which are difficult to handle, can be analyzed using GCXGC.