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INTRODUCTION TO CHROMATOGRAPHY

ERNST KENNDLER

Institute for Analytical Chemistry, University of Vienna

Ernst.Kenndler@univie.ac.at

Table of Contents

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THEORY OF CHROMATOGRAPHY..................................................................................................... 2 1.1 MIGRATION ............................................................................................................................................... 5 1.1.1 Migration velocity, capacity or retention factor ............................................................................. 5 1.1.2 Planar chromatography .................................................................................................................. 7 1.1.3 Retention time, tRi ............................................................................................................................ 8 1.2 DISPERSION ............................................................................................................................................... 9 1.2.1 Residence time distribution ............................................................................................................. 9 1.2.2 Plate height, plate number ............................................................................................................ 10 1.2.3 Packed columns............................................................................................................................. 11 1.2.4 Plate height vs. mobile phase velocity (van Deemter plot) ........................................................... 14 1.3 CHROMATOGRAPHIC RESOLUTION .......................................................................................................... 15 1.4 ADDITIVITY OF VARIANCES ..................................................................................................................... 18 1.5 DETECTORS: GENERAL CRITERIA OF PERFORMANCE ............................................................................... 19 1.5.1 Sensitivity ...................................................................................................................................... 20 1.5.2 Linearity ........................................................................................................................................ 21 1.5.3 Noise.............................................................................................................................................. 21 1.5.4 Instrumental limit of detection ...................................................................................................... 22 1.5.5 Specifity......................................................................................................................................... 22 1.5.6 Time response................................................................................................................................ 23 1.6 FURTHER READINGS ................................................................................................................................ 25

© Ernst Kenndler Version 19/01/2004

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Ernst Kenndler: Introduction to Chromatography

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THEORY OF CHROMATOGRAPHY

Separation of two sample components in chromatography is based on their different distribution between two non-miscible phases. The one, the stationary phase, a liquid or solid, is fixed in the system. The other, the mobile phase, a fluid, is streaming through the chromatographic system. In gas chromatography the mobile phase is a gas, in liquid chromatography it is a liquid.

Mobile Phase, m v

A

Stationary Phase, s Figure 1 Schematic presentation of a chromatographic system with partition of analyte A between the phases; v mobile phase velocity. The molecules of the analytes are distributed between the mobile and the stationary phase. When present in the stationary phase, they are retained, and are not moving through the system. In contrast, they migrate with the velocity, v, of the mobile phase when being there. Due to the different distribution of the particular analytes the mean residence time in the stationary phase differs, too, resulting in a different net migration velocity (see Figure 2). This is the principle of chromatographic separation.

Principle of chromatographic separation: Different distribution of the analytes between mobile and stationary phase results in different migration velocities

The position of the distribution equilibrium determines the migration velocity. It reflects the intermolecular interactions of the analyte with the stationary and the mobile phase. If only this process is considered, separation of the analytes as schematically shown in Figure 2 would be the result. 2

The analytes are inserted initially in a narrow zone. z Figure 2 Separation of analytes A. z. However. This broadening is greater the longer the migration distance is. respectively. in the time (t) or space (z) ordinate. B and C in time. B and C.Ernst Kenndler: Introduction to Chromatography A B C 0 t. t. or space. Dispersion is more pronounced as more 3 . Dispersion occurs around a mean value. They occur in parallel with a dilution of the initial sample zone with the mobile phase. In contrast. Two effects are therefore counteracting in the chromatographic system: the different migration is the base of separability by introducing separation selectivity. No zone broadening is taken into account. respectively. peak broadening effects lead to a potential overlap of the peaks migrating with different velocity. Partitioning of analyte molecules between mobile and stationary phase is a stochastic process. A B C 0 t. during migration of the analytes through the separation capillary broadening of the original sample zone takes place. z Figure 3 Depiction of a chromatogram of analytes A.

the chromatographic resolution. the following discussion will concentrate on linear analytical elution chromatography SYSTEMATIC OF CHROMATOGRAPHY state of phases gas / liquid gas / solid liquid / liquid liquid / solid Partition. 3. 1 surface packed open tubular thin layer paper mechanism geometry linear non-linear gradient isocratic 4 . The result is the formation of broadened analyte peaks.Ernst Kenndler: Introduction to Chromatography partitioning steps occur: thus it increases with increasing migration distance. From the methods used in practise (see Table about the systematic). leading to a chromatogram as schematically depicted in Figure. specific migration velocity and (ii) peak dispersion. absorption adsorption column planar amount of separand composition of mobile phase analytical preparative elution displacement GLC GSC LLC LSC 2 bulk phases 1 bulk phase. It should finally enable to express the extent of peak separation by a characteristic number. An appropriate theory of chromatography must be able to describe quantitatively the two counteracting phenomena: (i) the different.

1 1. Ki is the Nernst concentration distribution constant. in a small segment of the chromatographic system with length ∆z. V the phase volume.1 Migration Migration velocity. and cross section area As and Am.1. Substitution of the mole number gives 1 1 1 = = s s s ni 1 + k i` ci V 1+ m 1+ m m ni ci V (2) k` is the mass distribution coefficient s n is c is V k = m = m m = Ki q ni ci V . is given by n im n im + n is (1) where ni is the mole number of i. m. and q the phase ratio: 5 . being in average in the mobile phase. v m cim (t) Am cis (t) As z z+∆z s Figure 4 Schematic representation of a segment of the chromatographic system between z and z+∆z. i. The mole fraction of analyte. respectively. ci. i.Ernst Kenndler: Introduction to Chromatography 1. This concentration varies with time. capacity or retention factor We consider the concentration. i (3) Here c is the molar concentration. of an analyte.

of the separand i. It consists further from the concentration distribution constant Ki. which is in average in the mobile phase. and is given by eq. that is equal for all analytes for a given chromatographic system. ) (7) It can be seen that ui depends on the mean linear velocity. It was already mentioned that ki` consists of an unspecific part. it determines the extent of peak dispersion as well. s. ki`. The mass distribution coefficient. too. ki`. and on the mass distribution coefficient. When present in the mobile phase the analyte molecule migrate with velocity. Ki. capacity factor or capacity ratio. v. Contrary to liquid chromatography (LC) only one dimension exists thus in GC to influence the migration velocity of the analytes in a specific way for optimisation of the chromatographic selectivity. is of central importance for the description of the chromatographic migration. i. i. which is equal for all analytes the concentration distribution constant. Consequently the fraction of analyte. nevertheless. and thus the probability being there is (5) k i` 1 + k i` The migration velocity is therefore ui = k´ 1 v+ i ´ 0 1 + k i´ 1 + ki (6) or ui = v (1 + k i. 6 . v. ui. As in gas chromatography (GC) the mobile phase is considered as an ideal gas (that is inert by definition) no chemical interactions occur here. in which the specific chemical interactions between the analyte molecules and the stationary phase are reflected. that reflects the specific chemical interactions. This ratio is equal to the probability of one molecule being in average in the mobile phase. For the derivation of an expression for the migration velocity.Ernst Kenndler: Introduction to Chromatography c is Ki = m ci q= Vs Vm (4) It can be seen that ki` depends on the unspecific phase ratio q. 2. the phase ratio. ki` is named retention factor. being in average in the stationary phase. we ask for the relative amount of analyte.

relates the migration distance of an analyte with velocity ui to the solvent front R Fi ≡ zi z0 (8) With zi = ui t z 0 = vt (9) the RF value is R Fi = 1 1 + k i' (10) 7 . but decreases with time. The migration distance of a nonretained component is equal to that of the solvent front. 0 zi z0 Figure 5 Scheme of a planar chromatogram with three analytes The retention factor. Therefore the mobile phase velocity is not constant.Ernst Kenndler: Introduction to Chromatography 1. typically by capillary forces. differs. Sample components are separated by a solvent. z0 (see Figure 5). which is moving through the system. but their migration distance.1. After a certain time the development of the chromatogram is stopped.2 Planar chromatography In planar chromatography the separation system is a layer. zi. RF. This means that the migration time is equal for all components.

The retention time is given by t Ri = t Ro (1 + k i.1. which is identical with tR0. 8 . Transformation of eq. ) We can rewrite this equation as t Ri = t R 0 + t R 0 k i. and the migration velocity of the solute t Ri = L L(1 + k i.k´).3 Retention time. it is identical with the position of the peak maximum in case of a Gaussian elution profile. It is the k´-fold dead-time (tR0. which is the time the mobile phase needs to stream through the capillary column with an average linear velocity v measured over entire length. L. ) = ui v (11) with tR0 . This is equal for all components (and therefore non-specific). The second part is the solute-specific contribution on the total retention time. In the mobile phase the molecules exhibit the same velocity. With other words. This contribution depends on Ki. and consequently on the chemical interactions between analyte and stationary liquid.i − t R 0 tR0 (15) (14) (13) and can interpret it as following: the total retention time consists of two contributions ki´ can be calculated from the retention time of the analyte. that part which determines the separation selectivity. when differences in the partition constants are achieved. i. t Ri = t m + t s The one is tm. 13 leads to an expression that enables the experimental determination of the capacity factor: k i. tRi The retention time is the time in which halve of the quantity of a solute. and the residence time of a nonretained compound. the time the molecules are in mobile phase. L. is eluted from the chromatographic system. Separation selectivity can thus be established due to the selection of the phase system. the residence time of a non-retained component L = t R0 v (12) the dead time. It is determined by the length of the column.Ernst Kenndler: Introduction to Chromatography 1. = t R .

2. The residence time distribution function describes the concentration of an analyte in the mobile phase. z ) (16) The chromatographic column acts (under certain boundary. and that the peak width is negligible compared to the migration distance (σt << t) the resulting concentration partition function is given by a normal distribution. 1. Empirically. tR 9 .0 c max 0.2 Dispersion 1.4 0. t Figure 6 Gaussian concentration distribution function of an analyte with retention time. It is therefore the mean linear velocity <v>.or Dirac function). Under the conditions that the sample is initially brought into the system in an infinitely narrow zone (a so-called δ. and from the theory of chromatography it follows that zone broadening increases with increasing migration distance.1 Residence time distribution During the movement of the analytes through the separation column processes occur that lead to a broadening of the sample zone.6 2σ τ 0. which is to be used in the equations given above.e. i.Ernst Kenndler: Introduction to Chromatography In contrast to LC the mobile phase in GC is a compressible medium.8 concentration.2 0. c 0. averaged over the column length. as function of time and space. and the velocity of the mobile phase differs across the separation column: at the top of the column it is smaller than near the open end.and limiting conditions) as Gaussian operator on the concentration distribution. 1.0 0 50 100 150 200 250 tR time. either in the time domain or in the space domain: cim = f (t . cim.

i 2 (17) The maximum concentration is given by m c max. σt.i = ni 2π A σ t . The Gaussian bell curve is determined by three characteristic parameters: the position of the concentration maximum. by the standard deviation (here in the time domain). It gives the concentration of the component as function of time at a given position in the column (reasonably that of the detector after migration distance. increases with increasing migration distance.i . tRi the height in the maximum. The spatial distribution function (the concentration at a certain time as a function of the spatial coordinate) is of minor importance in column chromatography. is called plate height (height equivalent of a theoretical plate. σt .2. The Gaussian concentration partition function can be expressed as m cim = cmax. are related to each other by the migration velocity: 10 . ) m (18) Am is the cross sectional area of the mobile phase. H. the square of the standard deviation in the space domain. It follows from eq.i (1 + k i. It has relevance in planar chromatography. respectively. expressed e. and time domain. cmmax. σz.i e 1 t −t − Ri 2 σ t . which is directly proportional to the length of migration. 17 that the standard deviation (as a measure for the peak width) can be used as a central variable to describe peak dispersion 1. In fact it is the variance.Ernst Kenndler: Introduction to Chromatography The resulting function is depicted in Figure 6 in the time domain. (19) The factor of proportionality. the highest concentration of the analyte in the mobile phase the width of the function. σz . z σ 2 = Hz z HETP) and has the dimension of a length. Note that the variance in the space domain. L).2 Plate height. but underproportional related to the distance.i. plate number It is one of the central facts in chromatography that the spatial standard deviation. This increase is not proportional. σz2 .g. the retention time.

ϕ ) (24) which are listed in the following Table: 11 .i L = σ z . d c . which in sum determine peak dispersion: ⇒ ⇒ ⇒ ⇒ Hdiff describes the contribution from longitudinal diffusion Hconv that from convective mixing Hex.2. It is the result of 4 processes that occur during the migration of the analyte zone through the separation column. The smaller peak broadening is (after a certain distance) the smaller is the value of H.i = σ z2. d p . The total plate height is a function of the following experimental parameters and variables: H = f (v. d f . which incrementally contribute to the total plate height. N. k i´ .i u 2 i = Hi L H L H 2 = 2 i 2 = i t Ri 2 ui L t Ri L (20) The plate height.i 2 (23) 1. H.m that stemming from the kinetics of mass exchange from the mobile phase to the Hex. It is given by N= L H (22) In practice the plate number is determined from the peak in the chromatogram by 2 t N = Ri σ t .Ernst Kenndler: Introduction to Chromatography σ t2.s (21) It must be pointed out that the adequate measure for peak broadening which is relevant for peak separation is the plate number.m + H ex . D si . interface between mobile and stationary phase Consequently the total plate height is the sum of the four contributions: H = H diff + H conv + H ex . caused by the mixing of the analyte with the mobile phase. Therefore the plate height is composed from 4 incremental contributions. describes the extent of peak dispersion. Dmi .3 Packed columns It was pointed out above that peak broadening is the result of 4 processes. rather than the plate height.s that from the kinetics of mass exchange from the stationary phase.

it depends only on the diffusion coefficient. This time is proportional 1/v. and the time available for diffusion . Dmi .Ernst Kenndler: Introduction to Chromatography v Dmi Dsi ki´ dp dc df average velocity of mobile phase diffusion coefficient in mobile phase diffusion coefficient in stationary phase capacity or retention factor particle diameter of packing capillary diameter film thickness of stationary phase geometric factor for irregularity of packing ϕ The particular contributions are described in more detail in the following. 12 . which is the cause of a diffusional mass transport in the longitudinal direction according to 1st Fick`s law: dc dc = −D dt dz (25) cim z Figure 7 Longitudinal diffusion of sample zone in mobile phase. Hdiff The sample zone forms a concentration gradient in z direction. This diffusional mass transport is independent of partition and flow.

Hex. partitioning between the 13 .Ernst Kenndler: Introduction to Chromatography Hconv In a packed bed the channels between the particles have different size and direction. are inhomogene. The plate height increases with increasing mobile phase velocity. to the interface.s v zero mobile phas e stationary phase v mobile phase station ary ph ase Figure 8 Schematic presentation of the sample zone in the mobile and the stationary phase. For the (unrealistic) case that the fluid phase is not moving. Hex. Therefore both. dp. Note that this effect of peak broadening is diminished by transversal diffusion. the magnitude and direction of the local flow vector. The resulting peak broadening is independent of partition. respectively. Dmi.m . and depends on v. and the solute molecules migrate with different velocity at different positions in the column. Peak broadening in the flowing system occurs because there is time needed for the transport of the solute molecules from the mobile and the stationary phase.

B stems from longitudinal diffusion and C from mass exchange resistance. The dependence of the plate height on this velocity is described by the van Deemter equation. The kinetics of mass transfer is dependent on v. Therefore no additional peak broadening due to the finite kinetics of mass exchange would occur. ϕ. a dependence as shown in Figure 9 is observed 14 .4 Plate height vs. It increases with mobile phase velocity. in the practical case when the fluid phase is moved. Dmi. In summary in the following Table the influence of increasing values of the parameters v. Dsi . dp on the particular plate height contributions are listed = H decreases. because it enables to adjust separation efficiency.Ernst Kenndler: Introduction to Chromatography two phases has sufficient time to reach the stationary state. dp. Dsi. In practice in liquid and gas chromatography. Dmi . v Hdiff Hconv Hex. ki`. however. mobile phase velocity (van Deemter plot) One of the most important experimental variables is the mobile phase velocity.2. The situation changes. which reads in a simplified form as H = A + B v + Cv (26) The parameter A includes so-called eddy dispersion for packed columns. respectively. because then at the front of the peak there is an excess of solute molecules in the mobile phase.m Hex. at the rear side of the peak an excess is in the stationary phase.s Dmi = H increases Dsi dp - - 1. and the resulting peak broadens.

3 Chromatographic Resolution The characteristic number. vs.Ernst Kenndler: Introduction to Chromatography Figure 9 van Deemter plots for GC and LC: plate height. mobile phase flow rate It can be seen that a singular velocity exists where the plate height has a minimum. Rji. Right from the minimum efficiency is lost. Left from the minimum. but analysis is speeded up. is the resolution. 1. the singular velocity with highest efficiency should be adjusted. this higher velocity range might be chosen. The resolution is a measure for the distance of the peak maxima. 15 . but on a scale based on the widths of the peaks. If separation is sufficient. This distance is not measured in absolute units. which describes the extent of separation of the chromatographic peaks of a pair of solutes. H. in the area of pronounced diffusion. If separation is critical. i and j. efficiency is lost together with analysis time.

The generally accepted definition has the form (in the time domain) R ji ≡ t Rj − t Ri 2(σ t .i 2 σ t. which has a much larger potential for optimisation than the definition given by eq. 4 ( ) (28) αji is the selectivity coefficient defined as α ji = k 'j k i' It can be seen that the resolution consists of three terms: (29) 16 .i ) (27) It is clear that this definition is not very operational. tR Figure 10 Chromatographic resolution of the two peaks for components i and j.Ernst Kenndler: Introduction to Chromatography t R. For analytes with similar capacity factors (the analytical case of interest) the following expression for the resolution can be derived.j m igration tim e. and therefore this expression is transformed for practical reasons by substitution of the peak width by the plate height and the retention time by the capacity factors. 27: k.i t R.j 2 σ t. j + σ t . 1 N R ji = α ji − 1 1+ k .

6 0. The term approximates unit value at large k`.Ernst Kenndler: Introduction to Chromatography the selectivity term (αji -1). and the efficiency term.0 k / (1+k ) . especially when highly volatile solutes are to be separated. which may possess low k` values. The dependence of this term on the retention (or capacity) factor is shown in Figure 11.8 0. √N. or by the appropriate selection of the kind of the stationary phase or its film thickness. 6 8 10 Figure 11 Dependence of the retardation term in the resolution equation on the capacity factor. k`. It follows that it is of advantage to establish not too low k` values.g. It can be seen that the value of this term is small at low k`. In GC this can be done e. by decreasing the working temperature of the column. 1. and influences therefore the resolution unfavourably in this range. What is the significance of these three terms for the adjustment of the resolution? (i) The retardation term . . the retardation term k´/(1+k´). In LC it is adjusted by the selection of stationary and mobile phase as well. 17 .2 0. 0.having less importance in HPLC or in GC with packed bed columns – can be of interest in capillary GC.0 0 2 4 k .4 0.

that is only 40 %. 1. However. 18 . are found under the square root in the resolution equation only. σ2. In case of normal distribution.4 Additivity of variances It was seen that the total plate height generated due to the chromatographic process is the sum of the increments of the plate heights stemming from the individual contributions: longitudinal diffusion. In this case the selectivity coefficient αji is very close to unit. (iii) The selectivity term is that with the highest potential for optimisation. it is the square of the standard deviation. and elute close to each other. respectively. slow kinetics of mass exchange in the mobile and in the stationary phase. less than 5 %) can result in drastic changes of the selectivity term (which is α -1). This improvement in resolution must be paid. by the additivity of variances. especially in this case minute changes of the selectivity coefficients (e.1. this is 100 %). The variance is the second moment of the distribution function (the chromatographic peak). and therefore L. in the given example the term changes from 0.Ernst Kenndler: Introduction to Chromatography (ii) The third term of the resolution equation contains the plate number. enlargement of the column length increases N. with other words. which are crucial to separate. from 1. because they exhibit very similar capacity factors. In the same way the resolution is increased here strongly by a factor of 2.g. an increase of the column length by a factor of 2 leads only to an increase of the resolution by the factor of 1.10. This finding can be expressed also as the result of system theory. If the resolution is not sufficiently large. however. As N. by the double increase of the analysis time.4. This summation can be done because the chromatographic system behaves linearly.05 to 1. This holds especially for those pairs of analytes. because the processes responsible for the individual contributions are acting independently from each other.05 to 0. convective mixing.

exponential. dead Arbitrary.g. so-called extracolumn effects. Gaussian.Ernst Kenndler: Introduction to Chromatography The additivity of variance states that the total variance is the sum of the variances of the individual contributions (given that the system behaves linear. in many cases the variances from defined functions can be simply expressed analytically. volumes (tubings. 21 for the incremental addition of the individual contributions to band broadening. etc.m + σ ex . Extra-column Effects Sources Shapes finite injection zone. the additivity of variances also enables expressing plate heights for effects in addition to chromatographic ones being responsible for peak dispersion. s (32) Furthermore. detection volumes. Although not Gaussian shaped in general. e. Then the total variance is the sum: 2 2 2 σ tot = σ chrom + σ ex (33) These effects may stem from finite injection and detection volumes. tubings) Detectors: General criteria of performance We include here a discussion on general characteristics of detectors. rectangular.5 Width σ τ τ Variance σ2 τ2/12 τ2 Example Chromatographic column Injector.) triangular Function Gaussian Rectangular Exponential 1. although this topic is not limited to chromatographic detectors. 19) the additivity of the variances allows deriving eq. Properties of detectors are even not part of the theory of 19 . which leads to the equivalent expression (31) H tot = ∑ H ind For peak broadening in chromatography it reads 2 2 2 2 2 σ chrom = σ diff + σ con + σ ex . detector Mixing chamber (injector. from dead volumes of tubings. see above) 2 2 σ tot = ∑ σ ind (30) As the plate height is proportional to the variance (see eq. etc.

The factor of proportionality. In the linear range the sensitivity is constant. However. 20 . It should be noted that the detector sensitivity in a strict sense describes the change of the signal generated by a certain change of concentration or mass flow. Note that the sensitivity is not to be confused with the ability to determine small amounts of analyte. ai. c or m` curve (dashed line).1 Sensitivity S im m. dt (34) Concentration dependent detectors exhibit proportionality of the signal to concentration c S ic = a ic ci = a ic dni dV (35) 1. in contrast to concentration dependent ones. and independent of concentration. which are independent of dilution of the sample with mobile phase. in eqs. Outside the linear range it is still defined: it is the tangent on the S vs. Principally one can distinguish two detector types: (i) (ii) mass flow dependent and concentration dependent detectors. 34 and 35 is named the sensitivity of the detector a im = (36) or a ic = S ic ci (37) Mass flow dependent detectors give peak areas. detector characteristics are an important aspect in chromatographic practice. This is clearly indicated in Figure 12: the sensitivity is the slope of the calibration curve.Ernst Kenndler: Introduction to Chromatography chromatography in general. For mass flow dependent detectors the signal Si is proportional to the change of the mass m with time t S im = a im dmi = a im m .5.

m`. Si.Ernst Kenndler: Introduction to Chromatography S 20 .5. c or m` curve. Figure 12 Calibration curve showing the dependence of the signal.m ) 10 5 a = ∆ S / ∆ c. 0 0 5 10 15 20 c.2 Linearity The linear range is that concentration range where the sensitivity is constant and independent of concentration. Outside the linear range the sensitivity is still defined: it is the tangent on the S vs. c or m`) is linear. A large linear range is desirable. However. Note further that deviation from linearity does not necessarily hinder quantitative analysis. respectively.3 Noise The detector signal is always accompanied by a statistical fluctuation of the measuring values. the noise. 1.m . or its mass flow.g. It should be noted that at high concentration deviation from linearity (either convex or concave) is normal. of the analyte. c. In this range the calibration function (S vs. on the concentration. Due to its statistical nature the amplitude of the detection signal is Gaussian distributed. thermal or e. m . the availability of advanced calibration software in the last years reduced the importance of this parameter. This fluctuation has electronic. 21 . 1. The width of this distribution is typically characterised by the standard deviation. chemical reasons. 15 a = d S / d (c.5. σo (suffix 0 indicates the blank signal).

which is preselected according to statistical reasons.003 spike 0. E . If the value for b is e. The specifity is expressed for two substances i and j by 22 .5 Specifity The specifity.5. is that lowest concentration. zi. 1. this statistical certainty is 99. spikes and baseline drift.000 drift 0 2 4 6 8 10 12 14 time Figure 13 Chromatogram with noise.7 % (for 3 σo). the larger is the statistical certainty that a particular signal is not stemming from the noise.4 Instrumental limit of detection The limit of detection. It comes out that it is the relation of signal to noise (the signal to noise ratio) rather than the sensitivity that determines the ability of a detector to detect low amounts of analyte.Ernst Kenndler: Introduction to Chromatography 0.g. 1. which can be distinguished from the noise by a certain predefined probability. of a detector describes its ability to respond to different substances (at the same concentration or mass flow) with different signal values. to originate from the sample. in 3 cases it is mimicked from the noise. This means that in 1000 cases the signal is stemming 997 times from the sample. 3.5. with other words.001 6 σ0 0. The larger the value for b is taken. or smallest mass flow. zi = bσ 0 ai (38) b is that manifold of the standard deviation of the noise.002 spike signal 0.

but not the peak area (Figure 14). and quantitative analysis can be carried out most simply. i. b0. Experimentally the time constant can be 23 . in HPLC or in GC with packed columns.g.Ernst Kenndler: Introduction to Chromatography E ij = ai aj (39) Special relevance has the case where E is either 1 or ∞. Note that the property of the entire measuring unit is decisive. which.5 = width at halve height. because all quantities can be determined with one and the same calibration function. The detector is universal.6 Time response The time behaviour of the detector (including the units connected for data collection and processing) plays a more important role in capillary GC than e. In the extreme case E is infinitely large. Figure 14 Peak shape obtained with different time constants. The time property can be described by the time constant.5. does not occur in practice in general.e. For trace analysis the case is often most desirable that the detector response for matrix components is small. The time constant is a measure for the distortion of the profile of the measured values (the output signal of the detector) resulting from the given concentration profiles (the input into the detector). The time properties influence the peak shape. 1. τ. For E=1 all components are detected with equal sensitivity. however. τ . The peaks in the former method can be very narrow due to the high plate number reachable here. because then the specifity is large. and the time the peaks are passing the detector might be very short thus. only one single analyte is detected.

0 0. e.0 0 time Figure 15 Time response of detector. measured with the detector as function of time can be described for the case of a first order response by the function t − 1 − e τ c D = c0 (40) The time constant τ is the time the detector signal follows the concentration step to the value (1 − e )c −1 0 = 0.632 0.Ernst Kenndler: Introduction to Chromatography determined by the aid of certain measuring functions.g. cD .5 profile of m easuring value τ 0. by a rectangular concentration step of a sample added to the mobile phase. 24 . τ. concentration/co concentration profile 1. time constant A stepwise change of the concentration of a reference compound from zero to co occurs in the mobile phase at time t = 0. The concentration.632 c0 .

Kolb. Baars. Marcel Dekker. M. P. Hinshaw. G. Grob. 2nd ed. P. 1997. G. Huber. E. McNair. 1993. (12) Scott. Oxford. In Analytiker Taschenbuch.. 1997. W. Chromatography Today. Academic Press. Introduction to Analytical Gas Chromatography. D. Scott. Modern Practice of Gas Chromatography. 2nd ed. F. Detectors for Capillary Chromatography. S. Wiley. Schaller. Poole. Fehlersuche in der Gaschromatographie. L. 1999.. R. E. 2nd ed. H. W. Wiley. Jennings. 25 . Wiley. 1991. McMinn.. 1994. Hüthig. Wiley. Gas Chromatography. Marcel Dekker.. H.. J. E. H. K. 3rd ed. Hill. Basic Gas Chromatography. F. H.Ernst Kenndler: Introduction to Chromatography 1. (13) (14) (15) (16) Poole. I. Wiley. Elsevier. P. P. K. J. New York. Gas Chromatography: A Practical Approach. Fowlis. K. Advanstar. Introduction to Open Tubular Column Gas Chromatography. 2nd ed. B. 1994. Grant. Mittlefehldt. Analytical Gas Chromatography... R. 1998. Capillary Gas Chromatography. 2nd ed. Split and Splitless Injection in Capillary Gas Chromatography.6 Further readings (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) 1992. 1996. W. M. 1995. D... S. 1994. Grob. Ettre. Techniques and Practices of Chromatography. VCH. B. Kenndler. Baugh. Grob.... K. 1995... C. Hüthig. R. Springer. J. On-Column Injection in Capillary Gas Chromatography.. 1995. L. Gaschromatographie in Bildern. 1989. V. Miller. Stremple. W. 1991. 3rd ed. Wiley-VCH.

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