CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY

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1 Theme 06. CHROMATOGRAPHY (I): CHROMATOGRAPHY ELEMENTARY BASES 1599-ENVIRONMENTAL ANALYTIC METHODS Grade in Environmental Sciences Course Second Semester Professors: Miguel A Sogorb (msogorb@umh.es) y Jorge Estévez (jorge.estevez@umh.es) Department: Applied Biology CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. 1

INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS CHROMATOGRAPHY: Set of techniques for separating the components of a mixture on the basis of their different mobility in a porous medium (stationary phase)

2 INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS CHROMATOGRAPHY: Set of techniques for separating the components of a mixture on the basis of their different mobility in a porous medium (stationary phase) when they are carried by a fluid (mobile phase or eluent). Image taken from: net/intro/chromatogr aphy.html INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS STATIONARY PHASE A Mobile phase To the detector B Mobile phase To the detector C Mobile phase To the detector D Mobile phase To the detector 2

INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS CHROMATOGRAPHY: The name of chromatography (etymologically "Color writing") comes from initial experiments of Tswett, who separated diverse colored plant

3 INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS CHROMATOGRAPHY: The name of chromatography (etymologically "Color writing") comes from initial experiments of Tswett, who separated diverse colored plant pigments passing them through an adsorbent material content in a glass column. Animation about a chromatographic separation on open column: n.edu/analytical/animatio ns/lc_column.mov 1) Sample addition 2) Mobile phase addition 3) Separation by gravidity INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS 3

INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography.

4 INTRODUCTION TO CHROMATOGRAPHIC SEPARATIONS CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. 4

5 HISTORICAL DEVELOPMENT OF CHROMATOGRAPHY Background: coloring hair analysis. The discovery Tswett and plant pigments. Partition chromatography. Paper chromatography and thin layer chromatography. Gas chromatography. Exclusion chromatography. High-resolution liquid chromatography. Ion exchange chromatography. CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. 5

6 ELEMENTAL TERMS IN CHROMATOGRAPHY STATIONARY PHASE: Immobile material involved in the chromatographic process. The stationary phase can be a solid, a liquid, a liquid bound to a solid (partition chromatography) or chemical radicals bound to a solid (adsorption chromatography or ion exchange). SUPPORT: It is the material which sustains the stationary phase. The most common supports are columns and plates. MOBILE PHASE: Fluid that carries the sample through the solid phase. Mobile phase may be a liquid, a gas or a supercritical fluid. ELEMENTAL TERMS IN CHROMATOGRAPHY RETENTION: is the phenomenon which causes the immobilization of the analyte molecules due to the attraction between them and the adsorbent (stationary phase). ELUTION: is process by which an analyte is released from the adsorbent to which it was previously attached. ELUENT: Mobile phase used in the chromatographic process. The term eluent is reserved for liquid or supercritical fluid mobile phases (not for gas phases). ELUATE: Mobile phase exiting the column. 6

7 ELEMENTAL TERMS IN CHROMATOGRAPHY DETECTOR: A device that records a physic-chemical property of the eluate. CHROMATOGRAM: Representation of a physicochemical property of the eluate as a function of elution time or volume. Glossary of Liquid-Phase Separation Terms: 6/article.pdf A Compendium of GC Terms and Techniques: 00/article.pdf CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. 7

8 CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES MOBILE PHASE Liquid Gas Supercritical fluid CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES Column Plate Paper STATIONARY PHASE DISPOSITION 8

9 CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES Isocratic Gradient MOBILE PHASE COMPOSITION CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES Isotherm Gradient MOBILE PHASE TEMPERATURE 9

10 CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES ANALYTE-STATIONARY PHASE INTERACTIONS Adsorption Delivering Molecular exclusion Ionic exchange Affinity CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES ADSORPTION CHROMATOGRAPHY: Based on nonionic electrostatic interactions (dipole, hydrogen bonding, etc.). It can be divided into normal phase adsorption and reversed-phase adsorption. DELIVERING CHROMATOGRAPHY: The stationary phase is water retained in the pores of an inert solid matrix. The eluent is an organic fluid immiscible with water. More soluble analytes in the eluent are less retained by the stationary phase. 10

11 CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES IONIC EXCHANGE CHROMATOGRAPHY: The stationary phase is a matrix covalently attached to an inert solid having negatively or positively charged groups. Analytes with electrical charge of opposite sign of the electrical charge of the matrix will be retained and therefore separated from the rest of analytes with the same electrical charge than matrix and neutral analytes. AFFINITY CHROMATOGRAPHY: The interactions that occur between the stationary phase and analytes are quite specific between pairs of molecules: antigenantibody, hormone-receptor, avidin-biotin, antigenantibod, sugar-lectin, etc. CLASSIFICATION OF CHROMATOGRAPHIC TECHNIQUES MOLECULAR EXCLUSION CHROMATOGRAPHY: The stationary phase contains porous particles with a pore size comparable to the size of the molecules that are going to be separated. The separation is achieved because the analytes must travel different paths (shorter or longer according to their size) to reach the end of the column. 11

12 CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. CHROMATOGRAPHIC PARAMETERS: FOR INDIVIDUAL BANDS Dead volume (V O ) = Minimum volume of mobile phase that can carry a component of the sample along the entire column. Retention volume or elution volume (V R ) = Volume needed to elute a particular component of the sample. Corrected volume of retention or corrected volume of elution = Difference between the retention (or elution) volume and the dead volume. 12

13 CHROMATOGRAPHIC PARAMETERS: FOR INDIVIDUAL BANDS Dead time (t O ) = Minimum time needed for the mobile phase for carrying a component of the sample along the entire column. Retention time or elution time (t R ) = Time needed to elute a particular component of the sample. Corrected time of retention or corrected time of elution = Difference between the retention (or elution) time and the dead time. CHROMATOGRAPHIC PARAMETERS: FOR INDIVIDUAL BANDS 1,4 retención time (t R ) or retención volume (V R ) 1,2 1,0 signal 0,8 0,6 peak height = h peak width by half height (W 1/2 ) 0,4 0,2 peak width (W) half peak height = h 1/2 TIME (OR VOLUME) 13

14 CHROMATOGRAPHIC PARAMETERS: FOR INDIVIDUAL BANDS Peak width (W): Theoretical base of the triangle formed by joining the tangents to the inflection points of the Gaussian forming the peak. Peak width at half height (W 1/2 ): time or volume elapsed between half of the maximum height reached during the increase of the peak and half of the maximum height reached during the decrease of the peak. W = W 1/2 CHROMATOGRAPHIC PARAMETERS: FOR INDIVIDUAL BANDS 1,4 retención time (t R ) or retención volume (V R ) 1,2 1,0 signal 0,8 0,6 peak height = h peak width by half height (W 1/2 ) 0,4 0,2 peak width (W) half peak height = h 1/2 TIME (OR VOLUME) 14

15 CHROMATOGRAPHIC PARAMETERS: FOR INDIVIDUAL BANDS CAPACITY FACTOR (K): ratio between the corrected retention volume and the dead volume (or between the corrected retention time and dead time if the flow is constant). V R V 0 Capacity factor (k) = V 0 CAPACITY FACTOR IS NOT DEPENDENT OF SYSTEM GEOMETRY CAPACITY FACTOR IS DIMENSIONLESS CHROMATOGRAPHIC PARAMETERS: FOR INDIVIDUAL BANDS Parameter Mathematical expression Dead volume (time) V 0 (t 0 ) Retention volume (time) V R (t R ) Corrected volume (time) V R V 0 (t R t 0 ) of retention Capacity factor k = (V R V 0 )/V 0 = (t R t 0 )/t 0 Peak width at half height W 1/2 Peak width W = W 1/2 15

16 CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. CHROMATOGRAPHIC PARAMETERS: EFFICIENCY Chromatographic efficiency (N) is the parameter that quantifies the preference of the system for narrow bands. N = 16 (t Ri / W i ) 2 = 5.54 (t Ri / W 1/2 ) 2 CHROMATOGRAPHIC EFFICIENCY IS DIMENSIONLESS 16

17 CHROMATOGRAPHIC PARAMETERS: EFFICIENCY Chromatographic efficiency (N): Is a parameter for each single band of the chromatogram and thus not all the bands in the same chromatogram have the same chromatographic efficiency. Its utility is to compare chromatographic separations under different conditions. Is determined by factors such as the construction and packaging of the column and the mobile phase velocity. CHROMATOGRAPHIC PARAMETERS: EFFICIENCY Number of theoretical plate Mobile phase Stationary phase 17

18 CHROMATOGRAPHIC PARAMETERS: EFFICIENCY THEORETICAL EQUIVALENT PLATE HEIGHT (H): number of theoretical plates per unit length. H = L / N = (L / 16) x (W i / t Ri ) 2 The theoretical equivalent plate has units of length CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. 18

19 CHROMATOGRAPHIC PARAMETERS: PAIRS OF BANDS The separation factor (α 1/2 ) or specificity of two adjacent bands is defined as the ratio of corrected times of elution. 1/2 = (t R2 t 0 ) / (t R1 t 0 ) AND FOR CONSTANT FLOW 1/2 = (V R2 V 0 ) / (V R1 V 0 ) = k 2 / k 1 SEPARATION FACTOR IS DIMENSIONLESS CHROMATOGRAPHIC PARAMETERS: PAIRS OF BANDS CHARACTERISTICS OF THE SEPARATION FACTOR: The separation factor is always calculated with the band eluting later in the numerator. A high separation factor indicates that the centers of both peaks are far and thus the stationary phase retains one substance more than the other. Separation factor does not consider peak widths, which also determine the quality of separation. 19

20 CHROMATOGRAPHIC PARAMETERS: PAIRS OF BANDS RESOLUTION The resolution is determined by the stationary phase, mobile phase, temperature and length of the column. R S = 1 2 V R2 - V R1 x (W 1 + W 2 ) RESOLUTION FACTOR IS DIMENSIONLESS RESOLUTION IS ALWAYS ESTIMATED FOR OBTAINING A POSITIVE RESULT CHROMATOGRAPHIC PARAMETERS: PAIRS OF BANDS area 2,0 Resolution ,5 1,0 0,5 2,0 Resolution ,5 area 1,0 0,5 2,0 Resolution 0.80 RESOLUTION area 1,5 1,0 0,5 2,0 Resolution 1.0 1,5 area 1,0 0,5 2,0 Resolution ,5 area 1,0 0, TIME (min) 20

21 CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. PROBLEM ABOUT CHROMATOGRAPHY PARAMETERS ABSORBANCE 2,0 1,5 1,0 0,5 A 0, B TIME (min) PEAK C: t R =8min;V R = 0.6 ml/min x 8 min = 4.8 ml k B = (8-2)/2 = 3.0 W 1/2 = 1.2 min (0.72 ml) W = x W 1/2 = x 1.2 = 2.04 min (1.22 ml C DEAD TIME AND DEAD VOLUME: t 0 =2min; V 0 = 2 min x 0.6 ml/min = 1.2 ml PEAK B: t R =6min; V R = 0.6 ml/min x 6 min = 3.6 ml; k A = (6-2)/2 = 2 W 1/2 = 0.2 min (0.12 ml) W = x W 1/2 =1.698x0.2= 0.34 min (0.20 ml) 21

22 PROBLEM ABOUT CHROMATOGRAPHY PARAMETERS ABSORBANCE 2,0 1,5 1,0 0,5 A 0, B TIME (min) C ASSUMING THAT CHROMATOGRAM IS RUN WITH A 10 CM COLUMN LENGTH Peak B: N = 16 x (6/0.33) 2 = 5300; H = 10/5300 = cm Peak C: N = 16 x (8/2.04) 2 = 250; H= 10/250 = 0.04 cm The parameters N and H confirm what can be graphically seen. The peak B is much narrower and more suitable for chromatographic separation than peak C. PROBLEM ABOUT CHROMATOGRAPHY PARAMETERS ABSORBANCE B 2,0 1,5 C 1,0 0,5 A 0, TIME (min) Separation factor: 1/2 = (8-2) / (6-2) = 1.5 Resolution: 8-6 R s = = x ( ) 22

23 CHROMATOGRAPHY (I): BASIS OF ELEMENTAL CHROMATOGRAPHY INDEX: 1 Introduction to chromatographic separations. 2 Historical development of chromatography. 3 Elemental terms in chromatography. 4 Classification of chromatographic techniques. 5 Chromatographic parameters: o Individual bands. o Efficiency. o Pairs of bands. 6 Problem about chromatography parameters. 7 References. REFERENCES SOGORB MA y VILANOVA E. (2004). Introducción a la cromatografía. In: TÉCNICAS ANALÍTICAS DE CONTAMINANTES QUÍMICOS. Aplicaciones toxicológicas, medioambientales y alimentarias. Editorial Díaz de Santos. pp: ISBN: All pictures in this presentation, except when otherwise was indicated, were taken from this reference. 23

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