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).
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.
||UV Cut off (nm)
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.