Bioanalytical Chromatography Part I PDF

The 100-Year History of Separations Russian chemist and botanist Michael Tswett coined the term “chromatography” Chromatography was the first major “separation science” Tswett worked on the separation of plant pigments, published the first paper about it in 1903, and tested >100 stationa...

The 100-Year History of Separations Russian chemist and botanist Michael Tswett coined the term “chromatography” Chromatography was the first major “separation science” Tswett worked on the separation of plant pigments, published the first paper about it in 1903, and tested >100 stationary phases Separated chlorophyll pigments by their color using CaCO3 (chalk), a polar “stationary phase”, and petroleum ethers/ethanol/CS 2 Tswett’s original adsorption chromatography apparatus 1  History of Analytical Chromatography Chromatography was “rediscovered” by Kuhn in 1931, when its analytical significance was appreciated Chromatography very rapidly gained interest: Kuhn (Nobel prize in Chemistry 1937) separates caretenoids and vitamins 1938 and 1939: Karrier and Ruzicka, Nobel prizes in Chemistry R. Kuhn A. J. P. Martin 1940: established analytical technique 1948: A. Tiselius, Nobel prize for electrophoresis and adsorption 1952: A. J. P. Martin and R. L. M. Synge, Nobel prize for partition chromatography, develop plate theory 1950-1960: Golay and Van Deemter establish theory of GC and LC A. Tiselius R. L. M. Synge 1965: Instrumental HPLC developed 2  Overview of major milestones in the evolution of chromatography. The number of articles published in the literature according to PubMed servers and using the keyword chromatography are indicated in the top part of the figure. Legend: C – chromatography, GC – gas chromatography, GPC – gel permeation chromatography, HPLC – high performance liquid chromatography, IC – ionic chromatography, LC – liquid chromatography, MS – mass spectrometry, SFC – supercritical fluid chromatography, TLC – thin layer chromatography. CHROMATOGRAPHY  Chromatography operates as the same principle as extraction, but one phase is held in place while the other moves past it. Is the separation of a mixture based on the different degrees to which they interact with two separate material phases:  The two phases are: 1) The stationary phase: a phase that is fixed in place either in a column or in a planar surface. The stationary phase is either a porous solid used alone or coated with a stationary liquid phase 2) The mobile phase: a phase that moves over or through the stationary phase, carrying with it the analyte mixture. It is also called the eluting fluid. The mobile phase can be: i. a gas, ii. a liquid, or iii. a supercritical fluid. 4  Basic Classification of Chromatographic Methods Column Chromatography – stationary phase is held in a narrow tube through which mobile phase is forced under pressure Liquid chromatography – Mobile phase is a liquid solvent Gas chromatography – Mobile phase is a carrier gas Supercritical fluid chromatography – Mobile phase is a supercritical fluid Planar chromatography – stationary phase is supported on a flat plate or in the pores of a paper (e.g. TLC) 5  Modes of chromatography ❖The particles which fill a chromatographic column are not all the same hence the types of interactions between analytes and stationary phases are not all the same. ❖In liquid-solid chromatography the stationary phase is a solid such as calcium carbonate or more likely these days silica or alumina (Al2O3). ❖In gas-liquid chromatography the stationary phase is a low volatility liquid coated on to a solid support eg Carbowax 20M - a polyethylene glycol with a molecular mass of 20,000 g/mole. ❖In liquid-liquid chromatography, the stationary phase is a liquid, eg a C18 silane, covalently bonded to an inert silica support. ❖Movement between the stationary and mobile phase can be summarised by the following equation, where A is an analyte Amobile Astationary phase 6 Classification of chromatography according to the chromatographic bed shape and to the physical state of the mobile phase. The figure shows a solution containing solutes A and B placed on top of column packed with solid particles and filled with solvent (mobile phase). A separation technique based on the different rates of travel of solutes through a system composed of two phases - A stationary phase - A mobile phase Detect compounds emerging in column by changes in absorbance, voltage, current, etc Solute are separated in chromatography by their different interactions with the stationary phase and mobile phase Chromatogram (not spectrum) 8  Solutes which interact more strongly with the stationary phase take longer to pass through the column Strongly Retained Weakly Retained Solutes which only weakly interact with the stationary phase or have no interactions with it elute very quickly Fluid entering the column Schematic representation of a chromatographic separation. Solute A, with a greater affinity than solute B for the stationary phase, remains on the column Fluid emerging from the longer. end of the column 9 Results aimed for In any chromatographic separation, Detector response the following features are desirable: ▪ All analytes separated ▪ Well separated peaks ▪ Symmetrical, sharp peaks ▪ Short run times 10 minutes Retention time An example of a chromatogram Ideal results are not always easily achieved, however the following notes provide the basic information to enable simple chromatographic separations to be carried out. 10 According to the type of equilibration process involved, the chromatographic techniques can be classified as: The mode describes the way in which analytes interact with the stationary11phase. ADSORPTION CHROMATOGRAPHY - Solutes are separated based on their different abilities to adsorb to the support’s surface -- Uses an underivatized solid support (stationary phase = solid support) -- Oldest type of chromatography, but not commonly used If silica, a silicon-oxygen polymer as illustrated in figure is the stationary phase, the surface hydroxyl groups Si OH O interact with the analytes, the more the interaction the Si OH more the analytes are retained and therefore the longer O they take to pass through the column. Figure - structure of silica 12  Figure – binding of analytes to silica. Some analytes (pink) will bind more than others (blue) and hence separation is obtained. ❖The mobile phase (solvent) and analyte molecules compete for the adsorbent sites of the stationary phase. The more polar the molecules the longer they are retained. ❖Modification of the adsorbent sites can make it possible to separate isomers. ❖‘Strong solvents’ result in short retention times as the solvent molecules successfully compete for the stationary phase active sites which means there are fewer available sites to retain the analyte molecules. Figure – competition for adsorption 13 sites  Applications of adsorption chromatography ▪ Separation of relatively non-polar, water insoluble compounds ▪ Separation of isomers ▪ Purification of antibody fragments PARTITION CHROMATOGRAPHY Solutes are separated based on their different abilities to partition between the stationary phase and mobile phase. 14 In partition chromatography a solid support with a high surface area such as crushed firebrick or keiselguhr is coated with a high boiling liquid which acts as the stationary phase. Separation Solid support occurs because of the differences in solubility for the analytes in the stationary and mobile phases. The partition coefficient is defined as: Stationary phase Analyte Concn. in stationary phase Figure – coated support particle K = n Conc. in mobile phase 15 Applications of partition chromatography There are an immense number of possible applications of partition chromatography. The list below gives just a few examples of where this technique is routinely applied: ▪ Determination of water quality; ▪ Separation of aroma molecules of wine; ▪ Determination of pesticide residues; ▪ Quality control of pharmaceutical preparations; ▪ Identification and measurement on petroleum fractions. 16 BONDED PHASE CHROMATOGRAPHY In bonded phase chromatography, the molecule acting as the stationary phase is chemically bonded to the solid support. Si OH + Cl SiR Si O SiR + HCl R can be a C18 alkane chain or an amine (NH2) or cyano (CN) group or some other group. The nature of R determines the types of analytes which can be separated. The theories of partition and adsorption chromatography are both used to describe this mode of chromatography although it is often classified as a partition technique. Figure – diagram of phase bonded to silica 17 There are two types of partition chromatography normal phase and reversed phase, they are defined by the relative polarities of the mobile and stationary phases For this reason, the use of silica (a polar molecule) as the stationary phase (as in adsorption chromatography) is also considered to be a normal phase separation method. Because of its versatility and wide range of applicability, reversed-phased chromatography is the most frequently used HPLC method. 18  ION EXCHANGE CHROMATOGRAPHY As the name suggests, in ion exchange chromatography, ionic analytes are exchanged with ionic groups on the stationary phase - usually a polymer with ionic functional groups. ▪- Used to separate ions based on their different abilities to interact with the fixed exchange sites. The mobile phase is an ionic solution, such as a solution of sodium bicarbonate and carbonate (8 – 12 mmolar) for separating anions, or a sulphonic acid for separating cations. ▪- Uses a solid support containing fixed charges (exchange sites) on its surface ▪Cation-Exchange: support with negative groups ▪Anion-Exchange: support with positive groups 19 Applications of ion exchange chromatography ▪ Separation of vitamins ▪ Separation of inorganic cations and anions ▪ Separation low molecular weight organic acids ▪ Analysis of serum ▪ Analysis of drugs AFFINITY CHROMATOGRAPHY -- Separates molecules based on their different abilities to bind to the affinity ligand -- Uses a support that contains an immobilized biological molecule (affinity ligand) -- Commonly used to purify and analyze biological molecules -- Most Selective type of Chromatography 20 Affinity chromatography involves the interaction of a ligand (bound to a solid support with a spacer molecule) with the analyte. Figure - illustration of a ligand bound to the solid support There are two types of ligand that can be used in affinity chromatography. Those which are: ▪ specific and so bind to a specific analyte, and ▪ general and which bind to a group of analytes which have similar properties. A ligand acts in a similar way to an enzyme. 21 The five steps in affinity chromatography Affinity chromatography involves the interaction of a ligand (bound to a solid support with a spacer molecule) with the analyte. ▪ activation the ligand is bound to the chromatographic solid support. ▪ loading the analytes to be separated are introduced into the mobile phase stream. ▪ binding the analytes of interest are retained due to interaction with the ligand of the stationary phase. ▪ washing unwanted analytes are eluted from the column. ▪ elution the analyte(s) of interest are washed from the column by changing the mobile phase composition. These five steps are illustrated in figure shown on the next slide 22  activation load binding washing elution Figure – schematic diagram of affinity chromatography 23  Applications of affinity chromatography ▪ Purification of proteins ▪ Study of drug and hormone interactions with proteins ▪ Immunoassays SIZE EXCLUSION CHROMATOGRAPHY - - Separates large and small solute based on their different abilities to enter the pores of the support - - Uses a porous support that does not adsorb solutes - - Commonly used to separate biological molecules or polymers which differ by size (MW) 24 In size exclusion chromatography there are no chemical interactions with the mobile phase. The analytes are separated based on physical interactions - whether on not they are small enough to fit into the pores of the stationary phase. The stationary phases used are wide-pore silica gel, polysaccharides, and synthetic polymers like polyacrylamide or styrene-divinylbenzene copolymer. Pore sizes within the solid phases range from 4 – 250 nm. When an aqueous mobile phase is used, the technique is known as gel filtration chromatography (gfc sec) and the stationary phase will be hydrophilic (hydrophilic means water loving). If an organic mobile phase is used it is known as gel permeation chromatography (gpc sec) and the stationary phase will be hydrophobic (hydrophobic means water hating). 25 26 Application of size exclusion chromatography ▪ Determination of the molecular mass distribution of synthetic polymers ▪ Analysis of sugars ▪ Analysis and isolation of lipid polymers ▪ Purification, identification, and quantification of protein mixtures ▪ Study of polymer reaction kinetics ▪ Separation and purification of large biomolecules (molecular mass > 10,000 g/mole) 27 CHROMATOGRAPHIC TERMS 28 The chromatogram A chromatogram graph showing the detector response to analyte presence/concentration as a function of elution time (tR)A tM = dead time (a.k.a. t0) tR = retention time wB = peak width at base Non-retained solute (void volume) tM wB Point of injection ❖ Retention time (tr): the time it takes a compound to pass through a column ❖ Retention volume (Vr): volume of mobile phase needed to push solute through 29 the column The strength or degree with which a molecule is retained on the column can be measured using retention time or retention volume. FUNDAMENTAL MEASURES OF SOLUTE RETENTION Adjusted retention time (tr’): the additional time required for a solute to travel through a column beyond the time required for non-retained solute where: tm = minimum possible time for a non-retained t 'r = tr − tm solute to pass through the column Relative Retention (a): ratio of adjusted retention time between two solutes t'r 2 a= where: tr2’ > tr1’ , so a > 1 t'r1 -Greater the relative retention the greater the separation between two components 30  Retention time is dictated by physics and chemistry: – Chemistry (factors that influence distribution) stationary phase: type and properties mobile phase: composition and properties intermolecular forces temperature – Physics (flow, hydrodynamics) mobile phase velocity column dimensions The product of the tR and the eluent flow rate (F) is called the RETENTION VOLUME VR and represents the volume of the eluent passed through the column while eluting a particular analyte V R V= t =R Ft F R R 31 Capacity factor (k’): tr − tm The longer a component is retained by the column, the k' = greater the capacity factor tm Capacity factor of a standard can be used to monitor performance of a column Capacity factor is equivalent to: tim e s o lu te s p e n d s in s ta tio n a r y p h a s e Vs k' = k' = K tim e s o lu te s p e n d s in m o b ile p h a s e Vm where: Vs = volume of the stationary phase Vm = volume of the mobile phase K = partition coefficient Capacity factor is directly proportional to partition coefficient 32 The partition coefficient Once introduced into the chromatographic column, the analytes begin to distribute themselves between the stationary and the mobile phases in accordance with their partition coefficients. The partition coefficient (K) is defined as the ratio of these concentrations at equilibrium. Thus: Concentration of analyte in the stationary phase (Cs) K = Concentration of analyte in the mobile phase (Cm) Separation occurs between mixtures of analytes, when each analyte has a different ratio of solubility's in the mobile and stationary phases. 33  MOBILE PHASE VELOCITY The average linear velocity of analyte migration (in cm/s) through a column is obtained by dividing the length of the packed column (L) by the analyte’s retention time: L = L = length of column tR = retention time of analyte tR The average linear velocity of the mobile phase is just: L u= tM = retention time of mobile phase (“dead time”) tM Flow rate (mL/min) (F) is commonly used as an experimental parameter, it is related to the cross sectional area of the column and its porosity: F =  r u 0 2 u0 = linear velocity at column outlet  = fraction of column volume accessible to liquid 34  Retention and Differential Migration in Chromatography KA KB Distribution constant (partition ratio, partition coefficient): K A = c /cS A A M K B = c /cS B B M 35   Factors Affecting the Magnitude of the Distribution Coefficient (K) The magnitude of (K) is determined by the relative affinity of the solute for the two phases. Those solutes interacting more strongly with the stationary phase will exhibit a larger distribution coefficient and will be retained longer in the chromatographic system. Dispersion forces Dipole-Dipole Interactions MOLECULAR FORCES Polar forces Dipole-Induced- Dipole Interactions Ionic forces  All interactions between molecules are composites of these three forces. 36  Relationship Between Retention Time and Distribution Constant Need to convert distribution constants into something measurable – first express rate as a fraction of mobile phase velocity: m o le s o f s o lu te in m o b ile p h a s e average linear =u velocity of analyte to ta l m o le s o f s o lu te migration average linear velocity of MP cM VM 1 1 =u =u =u c M V M + c SV S 1 + c SV S / c M V M 1 + K VS /VM Substitute in definition of K The Retention Factor k: Then substitute L L 1 k= K VS =u 1 in definitions of =  VM 1+ k u and tR tM 1 + k 37  This leads to the definition k as the retention factor: L L 1 tR − tM V R − V M =  k = = tR tM 1 + k rearrange tM VM The more universal and fundamental retention parameter is the ratio of the retention volume to the dead volume The longer a component is retained tR VR known as the k = = by the column, the greater the tM VM capacity factor capacity factor k' Capacity factor of a standard can be used to monitor performance of a column Capacity factor is directly proportional to partition coefficient 38 k is a variable indicating how much time a component spends in the stationary phase compared to a non-retained inert component. k = 0; not retained k = 1; appears at 2 times to. k = 4; component spends 5 times to in the stationary phase The advantage of using the retention factor, rather than the retention time is the fact that it is independent of the column length and the flow rate of the mobile phase 39  Relative Migration Rates: The Selectivity Factor Selectivity factor (a): the ability of a given stationary phase to separate two components (t R ) B − t M t 'R ,B k B K B where: a = = = = t’R - adjusted retention time: the (t R ) A − t M t 'R , A k A K A additional time required for a solute to travel through a column beyond the time required for non-retained solute tM - minimum possible time for a non- retained solute to pass through the column t’R,B’ > t’R,A a is by definition > 1 (i.e. the numerator is always larger than the denominator) Different retention behaviour exhibited by the components 40  In general, if the selectivity of two components is equal to 1, then there is no way to separate them by improving the column efficiency. a is independent of the column efficiency; ▪ it only depends on the: ▪ nature of the components, ▪ eluent type, ▪ eluent composition, and ▪ adsorbent surface chemistry 41  Separation Efficiency and Peak Width The peak width is an indication of peak sharpness and, in general, an indication of the column efficiency. However, the peak width is dependent on a number of parameters : i. column length ii. flow rate iii. particle size In absence of the specific interactions or sample overloading, the chromatographic peak can be represented by a Gaussian curve with the standard deviation . The ratio of standard deviation to the peak retention time /tR is called the relative standard deviation, which is independent of the flow rate. 42  Efficiency of Separation The width of a solute peak is important in determining how well one solute is separated from another The peak width at the base (wB) is the distance between the intersections of the tangents drawn to the sides of the peak and the peak base geometrically produced. The peak width at the base is equivalent to four standard deviations (4) of the Gaussian curve and thus also has significance when dealing with chromatography theory. One measure of this is the width of the peak at half-height (w½ ) or at its baseline (wb) 43  ( t r2 − t r1 ) Rs = ( w b 2 + w b1 ) / 2 where: tr1 is the retention time of one analyte; tr2 is the retention time of the next analyte to elute wb1 and wb2 are the baseline widths of the peaks of analytes 1 and 2 at base. Note: both tr measurements are made from that of the unretained peak. ❑ Baseline resolution when R = 1.0 for triangles! ❑ Chromatographic peaks are not triangles they are Gaussian in shape; for Gaussian peaks Rs = 1.5 for baseline resolution (complete separation); ❑ When Rs = 1.0 only 94% resolution is obtained. 44  Good Resolution Chrom atogram 2.5 2 1.5 1 0.5 0 0 200 400 600 800 1000 1200 1400 Ti m e ( se c onds) Poor Resolution Chromatogram 2.5 2 1.5 1 0.5 0 0 200 400 600 800 1000 1200 1400 Ti me ( se c onds) 45  FACTORS FOR RESOLUTION  Two 2.5 Chrom atogram › The separation of the 2 peaks 1.5 1 › The widths of the peaks 0.5 0 0 200 400 600 800 1000 1200 1400 Ti m e ( se c onds) Chrom atogram  Both separations are the 2.5 2 same but the widths are 1.5 1 wider for the bottom 0.5 example. 0 0 200 400 600 Ti m e ( se c onds) 800 1000 1200 1400 46 ❖ The separation of two solutes in chromatography depends both on the width of the peaks and their degree of retention  Rs = Dtr / wave = 0.589Dtr/w1/2 ave ❖ The separation between the two solutes is given by their RESOLUTION (Rs) 47 48 COLUMN EFFICIENCY Plate and Rate Theories 49 was assumed to take place 50  6.) Measure of Column Efficiency Height Equivalent of a Theoretical Plate (H or HETP) -The distance along the column that corresponds to one “theoretical” separation step or plate (N) where: L = length of column N = number of theoretical plates H As H increases, more separation steps per column length are possible -Results in a narrower peak width and better separation between two neighboring solutes 51  In practice, it is more convenient to measure peak width either at the base line, or at the half height. 2  tr  2  t  N = 16   = 5. 55  r   w1   wb   2  52  tR 2 N = 16( ) wb tR tR wb wb Larger N Smaller N When the retention time, tR, is held constant, the column that produces peaks with narrower bases, wb, will be more efficient – have a greater N value. Likewise a column that produces wider peaks will be less efficient – have a smaller N value. This is because a smaller denominator, wb, will yield a larger overall number and a larger denominator will yield a smaller number. 53 Typical Plate Heights  GC ~0.1 to 1 mm  HPLC ~ 0.01 mm  CZE ~ 0.001 mm 54  Deficiencies of the plate theory The plate theory is far from perfect and, it has a number of limitations:  The plate theory assumes K is linear and therefore that the plates are symmetrical;  It assumes rapid equilibration of the analyte between mobile and stationary phases;  All peak broadening is not accounted for;  The effect of the mobile phase is ignored;  The dimensions (eg the thickness) of the phases are not taken into account 55  Chromatographic Rate theory Because of the limitations of THE PLATE THEORY it has been largely replaced by the ‘RATE THEORY assumed that there were three band spreading processes responsible for peak dispersion or peak broadening, namely: A B C Multi-path Longitudinal Resistence dispersion Difusion to mass transfer In 1956, Van Deemter introduced the first equation which combined all three sources and represented them as the dependence of the theoretical plate height (H) and the mobile phase linear velocity (u) 56  This is shown Van Deemter equation B H = A + + Cu linear flow rate u Multiple paths Longitudinal equilibration diffusion time L tM = retention time Remember: u= of mobile phase (“dead time”) tM A,B,C = constants for a given column and SP 57  H is affected by: Flow-rate of mobile phase Size of support: decrease size→ decrease H Diffusion of solute: increase diffusion → decrease H Strength of retention Others For a packed column the Van Deemter equation is directly applicable. However for capillary columns which are now commonly used in gas-liquid chromatography, a similar equation applies but without the ‘A’ term which is now zero, as capillary columns have no column packing. 58  Van Deemter “A” Term The “A” Term: Multiple Flow Paths or Eddy diffusion – As solute molecules travel through the column, some arrive at the end sooner then others simply due to the different path traveled around the support particles in the column that result in different travel distances. This differential flow of the solute molecules results in band dispersion. – molecules may travel unequal distances in a packed column bed – particles (if present) cause eddies and turbulence – “A” depends on size of stationary particles (small is best) and their packing “quality” (uniform is best) Molecules enter the Molecules exit the column column at the same time at different times due to different path lengths 59 The fact that the analyte molecules do not all take the same path through a packed column means that they do not all reach the detector at the same time hence peak broadening is observed. 60  Van Deemter “B” Term The “B” Term: Longitudinal diffusion – The concentration of analyte is less at the edges of the band than at the center. – The analyte diffuses out from the center to the edges. – If u is high or the diffusion constant of the analyte is low, the “B” term has less of a detrimental effect Narrow at point of introduction on the column Broadened analyte zone some time after introduction on to the column due to the diffusion of the analyte. 61  Van Deemter “B” Term The longitudinal diffusion (along the column long axis) leads to band broadening of the chromatographic zone. This process may be described by the equation: B Dm H = = 2 u u In this equation, Dm is the analyte diffusion coefficient in the mobile phase,  is a factor that is related to the diffusion restriction by the column packing (hindrance factor), and u is the flow velocity. – The higher the eluent velocity, the lower the diffusion effect on the band broadening – Molecular diffusion in the liquid phase is about five orders of magnitude lower than that in the gas phase, thus this effect is limited for LC, but important for GC 62  Narrow at point of introduction on the column Broadened analyte zone some time after introduction onto the Factors include: column due to the - Sample injection diffusion of the - Longitudinal diffusion analyte. - Finite equilibration between phases - Multiple flow paths - others 63 63  Van Deemter “C” Term Resistance to Mass Transfer: – The analyte takes a certain amount of time to equilibrate between the stationary phase and the mobile phase – If the velocity of the mobile phase is high, and an analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase – The band of analyte is broadened – The higher the velocity of the mobile phase, the worse the broadening becomes mobile phase movement off SP movement onto SP Stationary phase (SP) analyte attracted onto SP 64  Van Deemter “C” Term The C term is given by two parts (for MP and SP): f ( k ) d 2f f ' ( k ) d p2 H = C Su + C M u = u+ u DS DM where dp is the particle diameter, df is the thickness of the film, DM and DS are the diffusion coefficients of the analyte in the mobile/stationary phases, and u is the flow velocity The slower the velocity, the more uniformly analyte molecules may penetrate inside the particle, and the less the effect of different penetration on the efficiency. On the other hand, at the faster flow rates the elution distance between molecules with different penetration depths will be high. 65  Van Deemter plots ▪A plot of plate height vs. average linear velocity of mobile phase. The effect of the three terms in the van Deemter equation are shown graphically in figure Predicts that there will be an optimum velocity that gives a minimum value for (H) and thus, a maximum efficiency. Such plots are of considerable use in determining the optimum mobile phase flow rate. 66 Comparison between Plate and Rate theories The advantage of the RATE THEORY is that the terms A, B and C are defined in terms of experimental variables which in practise are seen to affect the chromatographic separation which is obtained. Typical variables are: ▪ Thickness of the stationary phase; ▪ Nature of the analyte; ▪ Nature of the mobile and stationary phases; ▪ Column length and diameter. The PLATE THEORY does not take into account all these variables nor does it take into account the mobile phase flow rate which as has been shown has a significant effect on the peaks obtained during a separation. However the plate theory does explain why separation is achieved and the shape of the peaks. 67  APPLICATIONS OF CHROMATOGRAPHY Chromatography is a powerful and versatile tool for separating closely related chemical species. ❑ In addition, it can be employed for the qualitative identification and quantitative determination of separated species. Important applications fields of chromatography in science development. 68 69 Co-occurrence network map related to the importance of chromatography in some fields of science.