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Optimising GC Temperature Programming

We want to share with you some tricks of the trade to enable you to understand and optimise your temperature programmed gas chromatography.

Scouting Gradient

A generic temperature program is usually employed with an unknown sample to investigate the nature of a sample (volatility range, number of components, suitability of phases etc.).  This is generally referred to as a scouting gradient.  A good general scouting gradient is shown (Figure 1).  The initial oven temperature should be as low as practically possible (usually 35 – 40 oC – this is usually dictated by the ambient temperature of the laboratory where the instrument is housed), the gradient rate is normally 10 oC/min. and should end at the column gradient maximum temperature (the higher maximum operating temperature as indicated on the column box or supplier specification) where a hold of at least 10 minutes should be employed.  This program will ensure that all analytes are eluted from the column and the relatively shallow temperature gradient should result in a reasonable separation of peaks.  This initial scouting run will likely be the longest run you make during any method development activity and the final method will be short shorter in the vast majority of cases.

Scouting Gradient

Figure 1: Generic scouting gradient used to investigate the retention behaviour of analytes in an unknown sample.

 

 
Isothermal or Temperature Programmed?

Following the use of a scouting gradient we can easily decide whether we need to use an isothermal or temperature programmed method by following a few quick rules of thumb.
 

1) If the peaks elute over less than 25% of the gradient time then isothermal operation should be possible.

For the scouting gradient shown:

tg = 27 mins.

ti = elution time of the first peak = 10.08 mins.

tf = elution time of last peak = 14.12 mins.

tr window = tf – ti  = 14.12 – 10.08 = 4.04 mins.

25% tg = 6.75 mins.

Therefore, all analytes elute over less than 25% of the gradient time and isothermal operation can be used.

2) If isothermal analysis is possible the Giddings approximation (Equation 1) can be used to predict the isothermal temperature.



Where:

T’ = isothermal temperature

Tf = elution temperature of the analyte of interest

See CHROMacademy for the full calculation

 
If isothermal elution is not possible a temperature programmed analysis must be carried out.  The parameters that should be optimised in temperature programmed GC are the initial temperature and hold time, the ramp rate, and the final temperature and hold time.  In order to simplify this process we have detailed some invaluable rules of thumb for optimising these parameters below
 

 

Initial Temperature & Hold Time

The initial temperature and hold time generally affect the resolution of early eluting peaks.  40 oC is often the lowest practical oven temperature that can be used without cryogenic cooling.  If there is a problem with the resolution of early eluting analytes the initial oven temperature should be reduced instead of adding an initial hold time (Figure 2).  As can be seen in Figure 2 decreasing the oven temperature from 100 oC to 40 oC increases resolution (Rs for critical pair 2,3 is 1.64 and 2.05 respectively). 
An increase in hold time from 1 – 2 mins. (at initial oven temperature of 50 oC) does improve resolution (Rs = 1.78) but not to the same extent as decreasing the oven temperature
.

 

Initial Temperature & Hold Time

 

Figure 2:  
Separation carried out with an

(a) initial oven temperature = 100 oC and hold time = 0 mins.
(b) initial oven temperature = 40 oC and hold time = 0 mins.
(c) initial oven temperature = 50 oC and hold time = 2 mins. 

Column: 5% Diphenyl dimethylpolysiloxane, 30 m x 0.25 mm x 0.5 µm. 
Inlet: Split 100:1, 1 µL, 180 oC. 
Flow: Constant linear velocity, 40 cm/sec. 
Carrier gas: Hydrogen. 
Detector: FID @ 250 oC. 
Gradient: 50 oC (no hold), 10 oC/min. to 300 oC, 20 mins. hold.

 
Let’s look at some tricks for optimising this parameter depending on your injection protocol.
 

 

Split Injection

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.

 

 

Ramp Rate

Adjusting the rate at which the temperature increases during the thermal gradient (ramp rate) has the most pronounced effect on the resolution of analytes which elute in the middle of the chromatogram.

We are often asked here at CHROMacademy what the optimum ramp rate is and the best advice we have ever been given was by the famous chromatographer Walt Jennings who once said that 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.


Therefore, if:

t0 = 30 s Ramp rate = 20 oC/min
t0 = 1 min. Ramp rate = 10 oC/min
t0 = 2 mins. Ramp rate = 5 oC/min


Once the optimum ramp rate for your system has been set it can be further optimised if required using ±5 oC/min. steps.  If higher or lower steps are used drastic or insignificant (respectively) changes may occur.

If there are a critical pair of peaks that cannot be resolved by altering or using multiple ramp rates follow the steps below.

  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.


  3. Start with a 1 minute hold and increase until separation occurs.
 

 

Final Temperature & Time

The final temperature should be determined from the scouting gradient analysis. So how do we decide on the final temperature for our separation?

If there are no analytes left to elute the final temperature should be set 10 – 30 oC above the elution temperature of the final analyte.

Therefore, if we have the following conditions:

Initial temperature (Ti) = 40 oC

Ramp rate = 10 oC/min

tf = 14.12 mins.

Then the analyte will elute at a temperature of:




Therefore, the final temperature should be in the range 191.2 – 211.2 oC.

If a thermal burn is required to remove late eluting analytes or stationary phase bleed and avoid possible carryover/contamination, a rapid gradient up to the gradient temperature maximum of the column should be included.  The hold time should be optimised until no more peaks are seen in the chromatogram.

 

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