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Theory and Instrumentation of GC

GC Temperature Programming

Aims and Objectives

Aims and Objectives

Aims

To describe and illustrate the relationship between temperature and retention in GC

To highlight the advantages and disadvantages of Isothermal and Temperature Programmed GC separations

To investigate the theory of Temperature Programmed GC and highlight several useful rules of thumb

To demonstrate the usefulness of Scouting Temperature Gradients for GC method development

To illustrate the development and optimization of a GC separation using a hypothetical scouting temperature gradient experiment

Objectives

At the end of this Section you should be able to:

Recognize the advantages and disadvantages of using temperature programmed GC

Explain how to implement a scouting temperature gradient for an unknown sample

Make decisions on whether samples can be run isothermally or need a temperature program

Suggest reasonable isothermal temperatures

Develop a temperature gradient for an unknown sample

Optimize the separation obtained from the temperature gradient suggested

© Crawford Scientific www.chromacademy.com

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Content The Role of Temperature in GC Separations 3 Isothermal and Gradient Temperature GC 4 Advantages of Temperature Programming 5 Disadvantages of Temperature Programming 5 Instrument Requirements 5 Theory of Temperature Programmed GC 6 Using and Developing Temperature Programs 7 Optimizing Temperature Programs 8 Initial Temperature and Hold Time 8 Adjusting the Ramp Rate 8 Final Temperature and Time 9


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The Role of Temperature in GC Separations Temperature is one of the two most important variables in GC and affects the retention (capacity (k)), selectivity (α) and to a lesser extent the efficiency (N) of all separations. As temperature is convenient to manipulate within a GC experiment, and as it has a large effect on the selectivity (and hence resolution) of a separation, it is a primary variable in method development and optimization. A general form of the Clausius-Clapeyron equation is shown opposite which links the analyte vapor pressure with the (inverse of) temperature. The equation indicates that as temperature is decreased, analyte vapor pressure decreases and hence retention time increases. This can be seen from the plot of log retention volume vs. 1/T.

Retention volume versus temperature

Clausius-Clapeyron Equation

ln (P

P°) =

∆H

R(

1

T°−

1

T)

Where: P = vapor pressure P° = vapor pressure at known temperature T° (K) ΔH = enthalpy of vaporization if the sample is a liquid or enthalpy of sublimation if it is a solid (Jmol-1) R = gas constant (8.314472 JK-1mol-1) T = temperature (K) Another form of the Clausius-Clapeyron equation is:


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lnP = −∆Hvap

RT+ C

ΔHvap = enthalpy pf vaporization (Jmol-1) C = constant There are several important conclusions that can be drawn from the nature of this curve:

As temperature decreases analyte retention (capacity) increases.

The lines are not parallel; therefore selectivity between analytes alters as a function of temperature.

As the lines diverge at lower temperature (towards the right of the plot), a useful generalization is that ‘GC separations are (usually) better at lower temperatures’.

Where the lines cross indicates co-elution – the two compounds cannot be separated at this temperature under these conditions.

As the plot lines cross this indicates a reversal in elution order, this is unusual but does occur. Method developers need to be very careful to ‘track’ peaks when developing temperature programs. Isothermal and Temperature Programmed GC In isothermal GC, the temperature of the GC oven (and hence the column) remains constant during the course of the analysis. This method of analysis is powerful when dealing with groups of compounds whose boiling points do not differ significantly and whose retention characteristics are similar. Where analyte retention characteristics differ problems can be encountered with long retention times, poor peak shape, and poor sensitivity for later eluting peaks; it may also be difficult to achieve a satisfactory separation for early eluting peaks where k < 2. In isothermal analysis the retention of peaks in a homologous series increases according to a logarithmic scale.

Isothermal analysis of paraffin (t=150oC) In isothermal analysis, when dealing with analytes whose retention behavior differs significantly, a fairly high temperature has to be used in order to achieve a reasonable analysis time. This has the disadvantage that poorly retained analytes elute with very low retention (k) and a reasonable separation may be difficult to obtain. When analyte retention behavior differs (usually due to a significant difference in boiling points); isothermal analysis can lead to increasingly longer retention times for each subsequent analyte. The retention behavior of subsequent analytes approximates to a


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logarithmic relationship, i.e. the time interval between peaks increases by a factor of 1.3 each time. Analytes with higher boiling points will show extended retention behavior which leads to broad peaks that show lower sensitivity (measured via signal to noise ratio). In this example, it appears that the last analyte in the column is C15 – this is shown to be untrue under gradient conditions.

Analysis of paraffin. Gradient temperature programmed analysis (50 to 250oC at 8oC/min) In temperature programmed GC, the oven starts at a low temperature to assist with the separation of early eluting peaks, it is then ramped and usually held for a specified time at an upper temperature. This allows the analytes to elute within a reasonable timeframe and ensures that all analytes have eluted from the column. In temperature programmed GC analytes of a homologous series will elute according to a linear scale. A useful benefit of gradient temperature programmed GC is that all peaks elute with approximately the same peak width. Later eluting peaks, therefore, do not suffer from dispersion as in isothermal GC and as they are ‘sharper’, the inherent sensitivity of the method (measured via signal to noise ratio), increases for later eluting analytes. Advantages of Temperature Programming

Useful for initial scouting runs to discover the nature of a sample

Better separation of compounds whose retention behavior differs

Improved detection limits, peak shape, and quantitative precision – especially for late eluting peaks

Enables column cleaning (or purging), by using a high temperature ‘burn’ at the end of the temperature program

Almost a requirement when using certain types of injection (i.e. splitless) in order to focus peaks at low initial oven temperatures

Disadvantages of Temperature Programming

More complex instrumentation with more variables to be optimized

Higher temperatures can give rise to rising baselines and increased noise (mitigated somewhat by using constant flow operation)

Phases suffer more rapid degradation through constant thermal cycling

The injection to injection time should be compared to isothermal analysis as the cool-down period may add a significant time to the analysis (especially when using low initial oven temperatures)

Instrument Requirements


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Dry carrier gas

Temperature programming capable oven

Electronic pressure control to enable constant flow operation is preferred

Suitable liquid phase that is capable of operating within the required temperature limits of the gradient

Theory of Temperature Programmed GC It has been demonstrated that an increase in oven temperature of 30 °C will reduce

analyte retention volume (time) by half. This also holds true for isothermal analysis and is useful for predicting retention changes when changing temperature. Therefore, in temperature programmed GC, the analyte rate of migration through the column will double for every 30 °C rise in the oven temperature. This is demonstrated by

the smooth curve in Figure 1.

Figure 1: Relative migration rate versus temperature In simple terms, the analyte moves through half of the column (volume) in the 30 °C prior

to elution (265 °C in this instance), three quarters of the column volume in the 60 °C prior

to elution etc. etc. This is true for all analytes; therefore, all analytes spend exactly the same length of time moving through the GC column in temperature programmed analysis. This is also the reason that all analytes have approximately the same peak width – they are dispersing within the column for the same period of time. Extrapolating back, all analytes are initially immobilized at the head of the column (focused), where they remain until the column temperature is suitable for them to begin to vaporize and partition through the column. Each analyte has a different relative vapor pressure, therefore, this is essentially the mechanism of separation in temperature programmed GC. Once the analyte begins to chromatograph, as explained above, it accelerates through the column at the same rate as all other analytes.


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Using and Developing Temperature Programs A generic temperature program is usually employed with an unknown sample to investigate the nature of the sample (volatility range, number of components, suitability of phase etc.) – this is known as a ‘scouting gradient’. A good general scouting gradient program is shown. The initial oven temperature should be as low as is practically possible within the laboratory (usually 35-40 °C); it should have a ramp rate of approximately 10 oC/min and

should end at the column gradient maximum temperature where a hold of at least 10 minutes should be employed. This program will ensure that all analytes within the sample are given an opportunity to elute from the column and the relatively shallow temperature gradient should result in a reasonable separation of peaks.

Single analyte

Multi analyte

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There are several useful rules of thumb that have been derived from the use of scouting gradients, these include:

If the peaks elute over less than 25% of the gradient time, then isothermal operation SHOULD be possible

If isothermal analysis is possible the Giddings approximation can be used to

predict the isothermal temperature: 45TT f , where T’ is the significant

(isothermal) temperature and Tf is the temperature at which the analyte of interest elutes from the GC column

An example of how this might be used in conjunction with a scouting gradient is considered within this section

Optimizing Temperature Programs Often, isothermal conditions are not satisfactory for the separation of a sample. Under these circumstances it is useful for the analyst to understand the important aspects of temperature programmed analysis and how to manipulate the program to optimize the separation. The information below indicates how one might improve the separation obtained from the scouting gradient. Initial Temperature and Hold Time The initial temperature and hold time generally affect the resolution of early eluting peaks within the chromatogram, with little to no effect on late eluting peaks. Adjusting the initial temperature has a greater effect on the early eluting peak resolution than adjusting the hold time. Decreasing the initial temperature will improve the resolution between early eluting peaks if required, however, the overall analysis time will be increased due to longer run and cool-down times. If the first peaks in the chromatogram do not elute for a significant time the initial temperature can be increased to save time. Care should be taken not to compromise resolution of early eluting peaks. Lowering the initial hold time can sometimes increase resolution between early eluting peaks without increasing analysis time, however, the effects are less noticeable than with initial temperature. Conversely, shortening the initial hold time can greatly improve peak shape and efficiency, sometimes also improving the resolution of early eluting peaks. It should be noted that extending the initial hold time past about 5 minutes can often lead to broad peaks and loss of resolution due to decreased efficiency. Split injection does not normally require an initial hold time as holding at a low initial temperature for a prolonged period will broaden peaks mitigating the efficiency (narrow analyte band) of the split injection. However, a caveat to this is that if you are operating at low initial oven temperatures (30 oC < solvent boiling point), which may be required to improve the retention of very volatile analytes, a hold time may be necessary. In this case start with 30 seconds and increase if necessary. Splitless Injection Splitless injection requires an initial hold time in order to ‘cold trap’ and/or ‘solvent focus’ the analytes. The hold time should be matched to the splitless time of the injection.


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Adjusting the Ramp Rate Adjusting the rate at which the temperature increases during the temperature program (the ‘ramp rate’), has the most pronounced effect on the resolution of compounds eluting in the middle of the chromatogram. Increasing the ramp rate has the effect of reducing the resolution between the mid-eluting analytes and reducing the overall analysis time. Reducing the ramp rate has the effect of increasing resolution between mid-eluting analytes, but increases the overall analysis time. Reducing the ramp rate will also increase the resolution between late eluting peaks but to a lesser extent. Use 5 °C/min. steps when altering the ramp rate as higher or lower steps may produce

drastic or insignificant changes respectively. It is possible to use multiple ramp rates to deal with difficult separations. Where areas of the chromatogram are cluttered and resolution is difficult to achieve a lower ramp rate may be used. Where areas of the same chromatogram are less cluttered, the ramp rate may be increased to save on overall analysis time.

The optimum ramp rate (in oC/min) is equal to 10 oC per void time (t0). Void time is equal to the retention time of an unretained compound.

Optimum ramp rate (℃/min) = 10 ℃

t0

For example:

Mid Ramp Holds Perhaps the ‘last resort’ in developing a gradient program is the use of a ‘mid-ramp hold’. Here an isocratic portion is inserted into the gradient to increase resolution between peaks in the middle of the chromatogram. It is best to begin the isocratic hold at a temperature 45 °C below the elution temperature of the first problematic peak (as per the Giddings

approximation) and to use a hold of between 2 and 5 minutes. This approach can be very effective but needs to be carefully optimized. Finally, with complex samples it may be necessary to use a mixture of all approaches which have been detailed.

The temperature at which to insert a mid-ramp hold can be determined using the following steps:

1. Determine the elution temperature of the critical pair.

2. Use Giddings approximation to determine the temperature at which to

include an isocratic hold, i.e. subtract 45 oC from the temperature at which

the critical pair elutes.

T′ = Tc − 45 ℃

t0 = 30 s t0 = 1 min t0 = 2 min

Ramp rate = 20 °C/min Ramp rate = 10 °C/min Ramp rate = 5 °C/min


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Where: T’ = isothermal temperature Tc = elution temperature of the critical peak pair 3. Start with a 1 minute hold and increase until separation occurs.

Peak Capacity Peak capacity (n) gives a measure of the number of components which can be separated

under a specific set of analytical conditions.1-2 Under isothermal elution conditions the

peak capacity is given by the following equation:

n = 1 + (N1/2

4) ln (

tn

ti)

Where:

N = column efficiency (H/L)

tn = retention time of final peak

ti = retention time of initial peak

Comparison of an isothermal and temperature programmed method demonstrates that

greater peak capacity can be achieved for temperature programmed methods due to al

peaks having the same peak width (Figure 1).

Figure 1: Representation of peak capacity for isothermal and temperature programmed

gas chromatography (GC).


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Therefore, the peak capacity for a temperature programmed method (n’) can be

calculated using the following equation.

n′ =N1/2

4[(

tn

ti) − 1]

It can be seen that using temperature programming increases the peak capacity of a

separation by a factor F of:

F =(

tnti

) − 1

ln (tnti

)

Example calculation

L = 30 m

H = 0.3 mm

ti = 50 s

tn = 1800 s

N = 100,000

For isothermal elution:

n = 1 + (N1/2

4) ln (

tn

ti)

= 1 + (1000001/2

4) ln (

1800

50)

n = 284 peaks

For temperature programmed elution:

n′ =N1/2

4[(

tn

ti) − 1]

=1000001/2

4[(

1800

50) − 1]

n′ = 2766 peaks

It can be seen that theoretically the peak capacity increases by a factor of 10 when

implementing a temperature programmed method.

For 2D GC (GCxGC) peak capacity is increased further to n1 x n2.

1. Giddings, J. C. Anal. Chem. 1967, 39, 1027-1028.

2. Horvath, C. G.; Lipsky, S. R. Anal. Chem. 1967, 39, 1893.


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Final Temperature and Time The final temperature should be determined from the scouting gradient analysis. If analytes continue to elute at the gradient maximum temperature then the temperature should be held at this point until all analytes have eluted. It is preferable, in general use, to have the gradient stop at the isothermal maximum temperature of the GC column and hold for longer to ensure all analytes are eluted. This makes the overall analysis time longer, however columns will last longer. The scouting gradient may indicate that it is unnecessary to increase the column temperature to the isothermal or gradient limit of the GC column. In this case the temperature should be raised approximately 10-30 oC higher than the elution temperature of the final analyte. Only include a hold at the upper temperature if programming to the column temperature limit – there is no reason to include a hold at the upper temperature of a temperature programmed analysis. The isothermal hold only acts to make analyte peaks broader and should be avoided. It is possible to carry out a thermal regeneration (or ‘burn’) of the column to ‘purge’ later eluting sample components which are of no interest, or highly adsorbed sample matrix components. This prevents fouling of the early part of the column and keeps it in good operating condition. This usually involves a rapid temperature gradient after the elution temperature of the last analyte of interest followed by a short hold time at the isothermal maximum temperature of the column. Be aware that this adds time to the overall analysis as oven cool-down times may be significantly longer.

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