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HPLC Gradient Elution – Baseline Drift

The CHROMacademy technical team was recently posed with the question of the causes of baseline drift in gradient HPLC (Figure 1).

basline drift

Figure 1: Positive (left) and negative (right) baseline drift.

The primary cause of baseline drift in gradient HPLC is due to changes in the refractive index of the eluent. During gradient elution the composition of the eluent will change and, hence, so will its refractive index. This usually manifests itself as a gradual increase in response during the gradient time. To compensate for the change in refractive index properties a reference wavelength should always be set otherwise drifting baselines will occur.

Positive baseline drift is commonly seen in gradient elution and is often caused by differences in the absorbance properties of the A and B solvents.  In the case of reversed phase HPLC gradient elution the eluent composition will change from a high percent of A (aqueous) to a high percent B (organic solvent).  The common organic solvents used in reversed phase HPLC have a greater UV absorbance (Table 1) than aqueous (water) which results in the rising baseline when monitoring at low wavelengths (~200 nm).  The problem of baseline drift caused by the disparity in UV absorbance can be remedied in three ways.

1) Absorbance matching

This involves the addition of a UV absorbing compound to the A solvent which increases its absorbance and, therefore, renders it equal to the absorbance of the B solvent, thus, eliminating baseline drift.    Any UV-absorbing compound can be used as long as it is unretained under the analytical conditions being employed and it does not react or interact with any of the analytes or matrix components.  Common compounds that can be used are inorganic ions, such as, nitrate, nitrite, azide, small organic ions, i.e. formate, acetate, and hydrophilic low molecular weight compounds, such as, urea, thiourea, or formamide.1-3 Determination of the correct concentration of UV-absorbing additive can be determined iteratively by addition of small amounts of compound and determining the effect on the observed baseline drift.

 
Solvent UV Cut off (nm)
Water 200
Methanol 205
Acetonitrile 190
THF 215

Table 1: UV cut off of common HPLC solvents.

The use of absorbance matching is not commonly used, but may happen without the realization of teh analyst due to the use of buffers as the aqeous component of the mobile phase (i.e. formate, acetate etc.).

A simpler and more routine solution to improving baseline drift is to use an alternative wavelength or a solvent which is transparent as the analytical wavelength.

2) Alternative Analytical Wavelengths & Transparent Solvents

As can be seen from Table 1 the UV cut off the common organic solvents which are utilized in reversed phase HPLC are in the range 190-215 nm.  Using these solvents and monitoring a low analytical wavelength will result in large baseline drifts in gradient analyses (Figure 2).  If the drift is very large and addition of a UV-absorbing modifier may not remedy the problem then an alternative analytical wavelength will be required or the use of a UV transparent solvent.

Figure 2: Comparison of baseline drift using methanol and acetonitrile.

 
 

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When possible, selection of an appropriate reference wavelength (i.e. with a diode array detector) will correct for solvent absorbance differences during gradient elution (Figure 3).

 

Figure 3: Comparison of an HPLC analysis carried out with (top) and without (bottom) a reference
wavelength »

 

Refernce wavelength


 

Another possible cause of baseline drift in HPLC gradient elution is poor column re-equilibration.  A drifting baseline will normally be seen during the re-equilibration phase of the gradient cycle (Figure 4), however, poor column re-equilibration may result in a persistent baseline drift which can also have adverse effects on chromatography.

 

Figure 4: Typical gradient HPLC profile »

 

Refernce wavelength


Equilibration - the time taken to ensure the whole of the analytical column is returned to initial gradient composition. This is an important step and if not properly considered can lead to retention time and quantitative variability.

The time required to completely re-equilibrate the analytical column prior to the next injection is dependent upon the column dimensions and the flow-rate used.  Most manufacturers recommend passing through ten column volumes of eluent at the gradient starting composition for complete re-equilibration, however, this can be determined empirically by shortening or lengthening the re-equilibration time and carefully observing any irreproducibility in retention times on successive injections of a test mixture.  Further, the equilibration time may be shortened by increasing the eluent flow-rate (take care not to exceed the maximum system operating pressure) during the equilibration phase, but care must be taken to ensure pressure stabilization at the original flow-rate prior to injection of the next sample.

Two important numbers are required to calculate the appropriate re-equilibration time are 1) the internal volume of your column (sometimes called the ‘interstitial volume’) and 2) the gradient dwell volume (the volume between the point of solvent mixing (usually in the mixing chamber or at the proportioning valves in the liquid chromatograph) and the head of the HPLC column). This value is particuarly important in gradient elution or when changes in solvent composition are made in isocratic elution so that the column experiences the composition change in the shortest possible time. Low pressure mixing systems tend to have larger dwell volumes than high-pressure mixing systems.

The term gradient dwell time will also often be used in relation to HPLC and is the time equivalent to the dwell volume which can be determined by multiplying the dwell volume by the flow rate. 

The column volume (Vm)can be calculated using equations 1 or 2.

Baseline Drift EQ 1 Where:
r = column radius (mm)
L = column length (mm)
For example, the volume of a 4.6 x 150 mm column will be:
Baseline Drift EQ 2


Therefore, at a flow rate of 1 mL/min, an equilibration of ten column volumes (17 mL) would mean allowing 17 minutes equilibration.

The gradient dwell volume is the total system volume in a gradient system between the point where the gradient is formed and the inlet of the column.  In a high pressure mixing system this includes the mixing chamber, connecting tubing, and injector.  In a low pressure mixing system this also includes the volumes of the pump head, pulse damper, and other connective tubing. System volume can be minimized by reducing tubing length and internal diameter, the number of unions used between tubing, using the correct column end fittings, reducing the injection loop volume, and reducing the detector flow cell volume.

A method for calculating dwell volume is described in interactive Figure 5.

 

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Figure 5: Calculating dwell volume.

 

A well plumbed LC system will have a dwell volume below 0.5 mL and some UPLC systems will be significantly lower.  However, if we err on the side of caution and assume 0.5 mL - this will add a further 0.5 minutes to the equilibration time at an eluent flow rate of 1 mL/min.

Therefore, a total of 17.5 minutes will be required to re-equilibrate this particular HPLC column and avoid problems with irreproducible retention times and separation selectivity.

 

References

  1. Snyder, L. R.; Kirkland, J. J.; Glajch, J. L.; Practical HPLC Method Development, Chapter 8.  Second Edition, John Wiley & Sons Inc. 2011.
  2. Berry, V. V. J. Chromatogr. 1982, 236, 279.
  3. Van Der Wal, S.; Snyder, L. R.  J. Chromatogr. 1983, 255, 463.

 

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