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Isocratic or Gradient?

You have been given a C18 column, an unknown sample and been asked to develop a reversed phase HPLC method.  Where do you start?  Do you use isocratic or gradient elution?  Don’t panic!  Let us make the decisions easy for you.

Using the steps below and the example scouting gradient (Figure 1) we will walk you through the decision making process, taking all the stress and worry away for you!

Step 1

Run a scouting gradient.  This allows you to investigate the elution behaviour of the analytes within the sample.  Typically we use a 5 – 100% gradient over around 20 minutes and examine the results to make some ‘rule of thumb’ predictions regarding the optimum mobile phase composition.  Typically it is not strictly necessary to control pH for neutral species; however, at lower mobile phase pH peak shape for polar compounds will improve.

scouting gradient

Hint
The use of 0.1% TFA as the aqueous eluent component results in a pH of ~2.1.  This will render most organic acids fully ion suppressed and most bases fully ionised.  This helps rationalise the results of the chromatogram in terms of analyte retention based on hydrophobicity and degree of ionisation.


« Figure 1: Example scouting gradient


Step 2

It’s decision time, isocratic or gradient elution.  Don’t panic, a good rule of thumb is


Isocratic analysis is possible when

Δtg < 0.25 tg

where: Δtg =(tf – ti)

tg = gradient time

So, for the scouting gradient shown in Figure 1 let’s see what elution method we should be using.

tf = 21.2 mins

ti = 12.8 mins

tg =20 mins

Therefore:

Δtg =(tf – ti)

       = 21.2 – 12.8

       = 8.4 mins

 

0.25 x tg = 0.25 x 20 = 5 mins

Δtg > 0.25 tg therefore, isocratic analysis is NOT possible.

 


Step 3

Gradient elution it is then, so how do we decide on our gradient conditions.  Gradients in HPLC are defined by three parameters: a) Initial %B, b) Final %B, c) and Gradient time (tg).  Another important parameter that needs to be taken in to account is the system dwell time (VD), this is the time taken from the gradient composition being mixed in the pump to the point at which this composition enters the column, and therefore, affects analyte retention.  This needs to be calculated for each system and details on doing this can be found in CHROMacademy.  For this example we will use 1.2 mins.

a) The elution composition of the first peak in our scouting gradient can be used to estimate the Initial %B.  In our example this would correspond to:

ti = 12.8 mins

VD = 1.2 mins

Retention time of first peak (ti) adjusted for system dwell = 11.6 mins

To estimate the initial %B we use the %B/min from our scouting gradient (i.e. gradient steepness)

Then calculate as follows:

%B/min x ti + starting gradient composition = 4.75 x 11.6 + 5 = 60.1 %B

b) The elution composition of the final peak in our scouting gradient can be used to estimate the Final %B.  In our example this would correspond to:

tf = 21.2 mins

VD = 1.2 mins

Retention time of first peak (tf) adjusted for system dwell = 20 mins

To estimate the initial %B we use the %B/min from our scouting gradient (i.e. gradient steepness)

Then calculate as follows:

%B/min x tf + starting gradient composition = 4.75 x 20 + 5 = 100 %B

c) Finally we need to decide upon the gradient steepness.  Gradient steepness is controlled by the mobile phase start and end composition and the gradient time.  The steepness of the mobile phase gradient can have a significant effect on the separation.

The equation for the gradient retention factor (k*) shown here can be found in many textbooks.  It can be rearranged to allow us to calculate the gradient time for our method.

tg = gradient time (min)

F = flow rate (mL/min)

S = constant determined by strong solvent and sample compound (Small molecules < 500 Da) the value is between 2 and 5; a value of 4 is used by convention when the value is not accurately known.  Proteins have much higher values (typically between 50 – 100) and need longer gradient times for separation.

ΔΦ = change in volume fraction of organic (Final %B – Initial %B)

VM = column void volume and is calculated using the equation below

k* = target value of 5 for average separation

Where: dc = column diameter (mm) and L = column length (mm).

For our example

Rearranging the equation so that we can calculate tg gives us



It is best practice to include an isocratic portion at the beginning of the gradient to allow transfer of our method to other HPLC systems.  Therefore, our initial gradient method for development is:

Time %B
0 60
1.2 (VD) 60 (Initial %B)
9.0 (tg+VD) 100 (Final %B

 

 

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