Theory and Instrumentation of GC Chromatographic Parameters i Wherever you see this symbol, it is important to access the on-line course as there is interactive material that cannot be fully shown in this reference manual.
Aims and Objectives Aims To introduce and explain the concept of Chromatographic Resolution (RS) To define the Resolution equation and illustrate its dependence on the chromatographic parameters Retention Factor (k), Selectivity ( α ) and Efficiency (N) To define Retention Factor (k), Selectivity (α) and Efficiency (N) in chromatography and show how each can be determined and optimised. To interactively demonstrate how each parameter may be manipulated to optimise chromatographic resolution Outline the fundamental basis for separation in GC To explain and demonstrate the concept of peak Asymmetry (A s ) Objectives At the end of this Section you should be able to: Estimate Resolution between peaks within a chromatogram Explain how Resolution is influenced by Retention Factor, Selectivity, and Efficiency Recognise which of these factors should be improved to increase Resolution within specific separations Develop strategies to efficiently optimise Resolution using the other chromatographic parameters Suggest ways to carry out optimisation and troubleshooting of separations in a practical context
Content Chromatographic Resolution (RS) 3 The Resolution Equation 5 Retention Factor (k) 6 How to Change Retention Factor (k) 8 Effects of Retention Factor on Resolution 10 Selectivity (Separation) Factor (α) 12 How to Change Selectivity (α) 14 Oven temperature. 15 Stationary phase 15 Effects of Separation Factor on Resolution 17 Efficiency 18 How to change Efficiency 21 Effect of Efficiency on Resolution 22 Peak Asymmetry 24 Crawford Scientific www.chromacademy.com 2
Chromatographic Resolution (R S ) The most important thing in GC is to obtain the optimum resolution in the minimum time. A resolution value of 1.5 or greater between two peaks will ensure that the sample components are well separated - to a degree at which the area or height of each peak may be accurately measured. In practical terms, a minimum resolution value of 1.7 is often applied to ensure reproducible separations. Resolution is calculated using the separation of two peaks in terms of their average peak width at the base (t R2 > t R1 ). Resolution calculation In the case of two adjacent peaks, it may be assumed that the peak width at the base w b1 w b2, and thus, the width of the second peak may be substituted for the average value. The width at the base of each peak is the segment of the peak base intercepted by the tangents drawn to the inflection points on either side of the peak as shown. It should be noted that as resolution increases so (generally) does the time required for the separation. As a chromatographer, you will have to balance the desire for rugged separations with that of time and materials. Crawford Scientific www.chromacademy.com 3
i Chromatograms at different resolution values You should be able to recognise baseline resolution and tell the difference between well resolved (R s >1.7) and poorly resolved (R s <1.7) peaks You should also have noted that analysis time increases as resolution increases you need more time to generate high quality separations. This needn t always be the case as will be demonstrated in later sections of the course Resolution can be reduced due to losses in efficiency (N) or selectivity (α). Resolution is also directly related to the retention factor (k). Above certain limiting values retention factor (around k=10) increasing retention will no longer result in an increase in resolution. This concept will be studied later on Crawford Scientific www.chromacademy.com 4
The Resolution Equation So how are we able to control the resolution obtained from a chromatographic separation? The Fundamental Resolution Equation (shown below), indicates that resolution is affected by three important parameters: Selectivity (separation factor) Efficiency Retention (capacity factor) So how do each of these factors contribute to the resolution of the separation and are there target values to aim for or limits beyond which a change in the parameter will have no meaningful effect? The answer to these questions is yes and it is important to understand the way in which each parameter affects resolution. The following pages will describe each of these parameters, how to change them and what affects they have on the resolution in Gas Chromatography. Capillary Gas Chromatography has inherently high efficiency, as we shall see later in this course. The relationship between the three parameters is shown: RS 1 N 4 1 1 Efficiency Selectivity Retention The fundamental resolution equation and its relationship to chromatographic parameters Crawford Scientific www.chromacademy.com 5
The next chromatograms represent two separations carried out using GC (1) and HPLC (2). The peaks in blue have the same selectivity value (α) and approximately the same retention factor (k). i Chromatographic parameters The HPLC chromatogram shows only moderate efficiency (N) the peaks of interest are relatively broad and are NOT baseline resolved (R s <1.5). The GC chromatogram shows high dfficiency (N) and therefore the peaks of interest are much narrower and are well resolved (R s >>1.5). In comparison GC is a more efficient technique however, HPLC is usually able to generate better separation selectivity. The primary driver for separation in GC is EFFICIENCY, the primary driver for separation in conventional HPLC is SELECTIVITY. Retention Factor (k) The retention (or capacity) factor is a means of measuring the retention of an analyte on the chromatographic column. The retention factor is equal to the ratio of retention time of the analyte on the column to the retention time of a non-retained compound. The non-retained compound has no affinity for the stationary phase and elutes with the solvent front at a time t 0, which is also known as the hold up time or dead time. Crawford Scientific www.chromacademy.com 6
There are several ways to determine t 0 including: The time at the baseline disturbance seen due to differences in differences in detector response to the injection solvent Retention time of Methane gas Retention time of Hexane (solvent) The Retention Factor is independent of some key variable factors including small mobile phase gas flow rate variations and column dimensions. Because of this, it is a useful parameter when comparing retention of chromatographic peaks obtained using different HPLC systems. Retention factor A high k value indicates that the sample is highly retained and has spent a significant amount of time interacting with the stationary phase. Chromatographers like to keep k values between 1 and 10 for good separations. If the t 0 time of the system was 1.0 minute, this would equate to a retention time rante of: t R ( k t ) t0 2.0 min( k 1) to 11.0 min( k 0 10) Crawford Scientific www.chromacademy.com 7
How to Change Retention Factor (k) The most effective and convenient way to alter the retention factor of a peak is to adjust the temperature of the mobile phase (carrier gas). This can be very simply achieved by entering the required temperature into the GC or data system. The instrument will then use its oven heater to regulate the column oven to the new required temperature. 155 o C i 175 o C Retention factor variation with temperature Column oven temperature effect. Conditions: Column DB1 30m 0.2mm 0.1µm. Flow 1.00mL/min, t 0 = 1.21 min. At low carrier gas (oven) temperature, the retention factor is high the analytes are interacting strongly with the stationary phase and their vapour pressure are low Increasing the oven temperature by 30 o C brings about a 2 fold reduction in retention factor (based on the Van t Hoff equation) When retention factors are very high or very low, the quality of the separation is reduced. Retention factors below 1 indicate that analytes may well be poorly resolved and may elute with the solvent peak and other non-retained coponents. High k values indicate the analyte will be excessively retained and peak shape will be poor and analysis times long Crawford Scientific www.chromacademy.com 8
Temperature is one of the two most important variables in GC, along with the chemical nature of the stationary phase. The retention factor (k) decreases as temperature increases they are inversely proportional - this is a fundamental relationship in Gas Chromatography. A decrease in vapour pressure (due to decreasing temperature (T)) results in a decrease in the relative amount of analyte in the mobile phase and an increase in retention factor (and retention time). This is described by the Clausius- Clapeyron and Van t Hoff equations. Important Clausius-Clapeyron Equation o H Log p ] Const 2. 3 RT Van t Hoff Equation dln[ k] H dt RT [ 2 Where: P o = analyte vapour pressure at a given absolute temperature T (K). ΔH = enthalpy of vaporisation at absolute temperature T (K). R = the gas constant (R = 8.314472 J mol -1 K -1 ) Retention volume versus temperature Crawford Scientific www.chromacademy.com 9
Log of retention volume (analogous to t r ) versus the inverse of temperature. The relationship between retention factor and temperature is clearly obvious from the linear nature of the plots for all analytes. The equations dealing with the temperature retention relationships in GC are complex as can be seen. In the practical complex it is enough to remember that retention is inversely proportional to temperature and that a plot of the natural logarithm of retention factor against the inverse of the temperature should give an approximately straight line relationship such as that shown The Clausisus-Clapeyron Equation shows us that as temperature decreases, the analyte vapour pressure also decreases (-1/T). As the analyte vapour pressure decreases it partition more readily into the stationary phase and is retarded in the column longer and hence it s retention factor and retention time increase The Van t Hoff Equation indicates that as temperature is increased the natural logarithm of the retention factor increases this is a direct proportionality between retention and temperature. We will study temperature retention relationships in GC in greater depth when we examine Temperature Programmed GC (ptgc) Effects of Retention Factor on Resolution The graphic below presents the effect of changing the retention factor on the resolution between two mid-eluting peaks in the chromatogram. i Resolution as a function of the retention factor Crawford Scientific www.chromacademy.com 10
i Resolution as a function of the temperature Crawford Scientific www.chromacademy.com 11
Note that the resolution between some peaks is affected more than others. This relates to the isotherm plots seen on the previous page if the plots are parallel resolution between the peaks is not greatly affected by temperature if the lines converge, diverge or cross then altering the oven temperature will have a larger effect on resolution. The largest gain in resolution is achieved when the k value is between about 1 and 10. k values less than 1 are unreliable as analytes may be eluting with other sample components or solvent. Above a k value of approximately 10, increasing retention only provides minimal increases in resolution. Too much retention wastes valuable analysis time and the chromatographic peak height will decrease as the bandwidth of the peaks increases. Some important considerations regarding retention factor are: For more complex sample mixtures, the useful k range may be extended to 2 < k< 20 Above a retemtopm factor of around 10, any increase in k makes little difference to the resolution the analytical run time also begins to get prohibitive If you have not achieved the desired resolution and the k values of your sample components are above 10, you will find that increasing the selectivity or efficiency of your separation will be more useful At very low values of k (<1.0) it is often difficult to obtain a meaningful separation. As the retention factor is very simple to change it is often worthwhile adjusting the k range of peaks within the chromatogram to obtain an optimum resolution Retention factor in Gas Chromatography is normally adjusted using the oven temperature, chemical nature of the stationary phase ro the phase ratio (ß) Selectivity (Separation) Factor (α) The selectivity (or separation factor) (α) is the ability of the chromatographic system to chemically distinguish between sample components. It is usually measured as a ratio of the capacity (retention) factors of the two peaks in question and can be visualised as the distance between the apices of the two peaks. By definition, the selectivity between separated peaks is always greater than one as when α is equal to one, the two peaks are co-eluting (i.e. their capacity factor values would be identical and there is no separation). The greater the selectivity value, the further apart the apex of the two peaks becomes. Selectivity values between the peak of interest and the PROCEEDING peak are quoted so if peaks swap over retention order there may be a sharp change in selectivity value. In contrast to HPLC, the options for altering the selectivity of the separation are somewhat more limited. As the selectivity is dictated by the chemistry of the analyte, the column temperature and the phase ratio (β) the stationary phase chemistry, oven temperature and the column internal diameter to film thickness ratio are commonly used to affect selectivity. Crawford Scientific www.chromacademy.com 12
Selectivity k k 2 1 t t R R 2 1 t t o o High alpha values (>1.0) indicate a good separation power and a good separation between the APEX of each peak. However, the alpha value is NOT directly indicative of the resolution. i Critical pair separation Crawford Scientific www.chromacademy.com 13
Other variables that affect the retention factor include the chemical nature of the stationary phase as well as the ratio between the amounts of carrier (mobile phase) and stationary phase inside the GC column. This latter parameter is often called the Phase Ratio (β) and will be discussed later. These latter parameters involve changing the GC column and are therefore less convenient than temperature for changing retention factor. How to Change Selectivity (α) In Gas Chromatography the choices for altering selectivity are more limited than with HPLC, however as GC is such a highly efficient separation technique, the requirements to adjust selectivity to optimise the separation are often lessened. Adjusting temperature to alter separation selectivity can often be unpredictable and some practical work is required to assess selectivity changes. This is also true of changing the temperature program ramp rate, however both of these changes are convenient and easy to make often resulting in faster method optimisation. Other changes to separation selectivity often require a change in column hardware either to a new stationary phase type or to an alternative phase ratio (column internal diameter to film thickness ratio). Whilst these changes are not so convenient, they often result in the most predictable and effective changes to separation selectivity both will studied in greater detail in subsequent course sections. Some of the factors that can be used to manipulate the selectivity of GC separations are shown in the table below. Table 1. Factors affecting selectivity Parameter Usage Oven temperature Ramp Will alter selectivity for some analytes less predictable but rate highly convenient. Oven temperature Increasing or decreasing the oven temperature will cause the relative band spacing to change less predictable than other parameters. Stationary phase Changing the stationary phase chemistry is the most powerful way to alter selectivity in GC analysis. Phase ratio (ß) The ratio of carrier gas to stationary phase alters the distribution constant of analyte molecules affecting the selectivity of a separation. The next figure presents the temperature effect on selectivity. 1.0 o C/min 2.5 o C/min i Selectivity as a function of temperature Crawford Scientific www.chromacademy.com 14
Oven temperature. We saw how temperature can be used to alter the retention factor (k) of a separation. You may have noticed that as the temperature changes the relative spacing (or selectivity) between some of the peaks also changes. i Temperature effect on retention It is clear that the selectivity of the peaks in the region highlighted has changed markedly. Examining the temperature retention plots remind us that when analyte isotherms are not-parallel, then altering the oven temperature will have an effect on the selectivity of the separation. The use of temperature to affect selectivity is powerful for some analyte components and less powerful for others thus whilst it is a highly convenient way to optimise selectivity, it is perhaps one of the less predictable methods, unless separations are undertaken to plot the temperature retention relationships for the various analytes. Stationary phase Changing the chemical nature of the stationary phase is amongst the most powerful ways to alter analyte selectivity in Gas Chromatography. The chemical interactions between the analyte and stationary phase include: dispersion, polar, hydrogen bonding, etc. and altering the nature of these interactions affects different analytes to different extents hence their relative retardation by stationary phase changes and selectivity is altered. This topic will be studied in greater depth later on. In the next figure we can see a large change in selectivity between two alkanes and two alcohols. Column 30m 0.25mm I.D. 0.25µm. Carrier gas: Helium ar 32 cm/sec. Oven: 50-110 o C at 5 o C/min. Crawford Scientific www.chromacademy.com 15
i Selectivity alteration by the stationary phase selection DB1 is a non-polar stationary phase (100% polydimethylsiloxane) and DB35 contains 35% phenyl functional groups making it more polar. The alcohols (polar) are retained more strongly on the second column RELATIVE to the alkanes and so selectivity is altered. Separation o different stationary phases Crawford Scientific www.chromacademy.com 16
The phase ratio describes the relative amounts of stationary phase and mobile phase within the column and is controlled by adjusting column internal diameter and stationary phase film thickness. Increasing the phase ratio makes it increasingly likely that an analyte will interact with the stationary phase so altering its distribution constant. As the distribution of the various analytes changes to different extent for each analyte the RELATIVE retardation of the analytes is altered and the separation selectivity changes. Capillary column Effects of Separation Factor on Resolution Use the slider to investigate the effect of changing the selectivity (separation factor) on the resolution between two early-eluting peaks in the chromatogram. In this example the selectivity has been altered using the temperature program ramp rate as in the previous page, but this time with a slightly different temperature program. It can be seen that as the selectivity rises above 1.0, the resolution improves dramatically. Altering the system selectivity provides an excellent means of optimising the chromatographic resolution as small changes in selectivity can lead to large changes in resolution. This can be seen from the selectivity / resolution curve opposite. A 4% increase in selectivity results in a resolution increase of over one order of magnitude excellent chromatographic value for money! Remember RS 1 N 4 1 1 Efficiency Selectivity Retention Crawford Scientific www.chromacademy.com 17
i 3.25 o C/min 0.25 o C/min Resolution as a function of selectivity Important Changing the selectivity can have a dramatic effect on the chromatographic resolution As capillary GC is a highly efficient technique, small changes in selectivity have a big impact on resolution as can be seen from the resolution/selectivity curve. An increase of 4% in the separation selectivity of the peaks of interest results in an order of magnitude increase in resolution There are several ways to alter the selectivity of a separation in GC including altering: oven temperature / temperature program ramp rate / column stationary phase / column phase ratio (ß) Temperature and ramp rate are normally adjusted first for convenience. If a suitable separation cannot be obtained the stationary phase or phase ratio are changed Efficiency The efficiency of a chromatographic peak is a measure of the dispersion of the analyte band as it travelled through the GC injection system, column and detector. In an ideal world, chromatographic peaks would be pencil thin lines however, due to dispersion effects the peaks take on their familiar Guassian shape. The plate number (N) is primarily a measure of the peak dispersion in the GC column, reflecting the column performance. N is derived from an analogy of Martyn and Synge who likened column efficiency to fractional distillation, where the column is divided into Theoretical Plates. Crawford Scientific www.chromacademy.com 18
Parameters Each plate is the distance over which the sample components achieve one equilibration between the stationary and mobile phase in the column. Therefore, the more ( theoretical ) plates available within a column, the more equilibrations possible and the better quality the separation. The more traps or plates there are, the narrower the carbon number range collected from that trap. Therefore, the higher the number of plates (N) the narrower the carbon distribution obtained from that trap. This concept can be directly related to the peak eficiency in GC where a column with a high number of plates gives rise to narrower peaks. Similarly, for a fractioning tower of a given length (L), the higher the number of plates, the lower will be the distance between each plate, shown as plate height in the diagram. Therefore, for high efficiency separations, the plate number (N) will be high and the plate height low. Note that plate height is often called Height Equivalent to a Theoretical Plate (HETP). Crawford Scientific www.chromacademy.com 19
i Distillation column working principle GC analogy These two terms are related through the expression: H = L / N Crawford Scientific www.chromacademy.com 20
The number of theoretical plates is often used to establish the efficiency of a column for a given method. The method developer may decide that a given method is no longer valid when the plate number falls below a predetermined value at which time the column would be replaced. Some typical column efficiencies for capillary GC are presented in the next table. Figures are plates (N) / meter (m) and plates (N) / column for a standard 30m column. The phase used was polydimethylsiloxane (PDMS) a popular non-polar stationary phase. Table 2. Capillary column efficiencies Column I.D. (mm) N/m (N/column) 0.10 10,000(300,000) 0.20 4,500(135,000) 0.32 3,200(96,000) 0.53 1,500(45,000) It is interesting to compare these figures with the plate count for a typical HPLC column (150mm 4.6mm 5µm) of between 5000 and 8000 plates per column. GC separations are primarily driven by the efficiency of the columns used therefore it is important to maintain high efficiencies where possible. How to change Efficiency There are many factors that contribute to the broadening of the peak or actually the injected band of analyte vapour as it travels through the chromatographic system. The biggest contributor to band broadening (and hence lower efficiency) is usually the column itself. The column length, whether it is a packed or capillary column, the film thickness, internal diameter and quality of column packing or coating all play a part in determining the column efficiency. Several other factors also need to be taken into account including: Injection volume. Dead volumes (in the injector or detector especially the column couplings). Flow rate. Type of carrier gas used. The relationship of efficiency to column length can be found in the next figure. Crawford Scientific www.chromacademy.com 21
i 10m column 120m column Separation of pesticides. Efficiency as a function of column length As column length increases the peaks become narrower (more efficient) As the peak efficiency increases the separation quality increases As the column length is increased, the analysis time increases significantly (this should be intuitive). Approximately speaking doubling column efficiency requires a doubling of column length and a doubling analysis time! Increasing the column length by an order of magnitude (for example 1m to 10m or 10m to 100m) the efficiency of the peaks of the peaks also increases by about one order of magnitude 100,000-1500,000 plates can be generated by a good 30m 0.2cm column with a reasonable thin film (0.1-0.5µm) It is important to understand that in GC, column length is one of the least important methods of increasing column efficiency column internal diameter and film thickness should be adjusted first to increase efficiency. Flow rate may also be adjusted to increase the separation efficiency Effect of Efficiency on Resolution In the next example (separation of pesticides) the efficiency has been altered using the column length. It can be seen that the increase in resolution follows an approximately straight line as efficiency is increased. Crawford Scientific www.chromacademy.com 22
i 10m column 120m column Efficiency as a function of column length The slope of this line is important. It should be noted that doubling the column efficiency, which will also mean doubling analysis time and cost of the column, will only increase the resolution by a factor of the square root of 2 (i.e. 1.42). It is doubtful if this would be considered good chromatographic value for money! As you investigate the relationship between efficiency and resolution, it s important to look at the retention time scale and notice the overall time for the separation. In this example, efficiency increases as column length increases It should be obvious that altering column length is able to provide base line resolution between the peaks of interest Whilst resolution is generally improved the transition from a column length of 10m to a 100m increases the analysis time by a factor of 10 (one order of magnitude). An important general rule of thumb states: Doubling length, doubles efficiency, doubles analysis time (and probably column cost) but only increases resolution by a factor of 1.42 times Altering column length is perhaps the least important way of increasing efficiency (and therefore resolution) in Gas Chromatography Before column length is increased, the column internal diameter and phase ratio may be optimised. Changing the carrier gas flow rate and the carrier gas used are also useful ways of improving resolution Crawford Scientific www.chromacademy.com 23
Peak Asymmetry In the ideal world all chromatographic peaks would be symmetrical. However due to the effects of instrument dead-volume, amount of analyte introduced onto the column (loading), adsorptive effects of the stationary phase and instrument components, peaks may often show a tailing or fronting behaviour. Tailing describes a peak whose tail portion (distance B in the diagram) is wider that the front portion (distance A in the diagram). Fronting is the reverse of the situation and the front portion is wider than the peak tail (A>B). Chromatographic peak description None symmetrical (asymmetric) peaks present problems with obtaining a suitable chromatographic resolution and quantitation of the peaks within the chromatogram. Asymmetrical peaks are more difficult to resolve and integration of the peak to provide area for quantitation will also be much less reproducible. Often, chromatographers will set limits for peak asymmetry beyond which chromatography will be deemed unsuitable. Chromatographic peak shapes Crawford Scientific www.chromacademy.com 24