HPLC Column Efficiency

Column efficiency refers to the performance of the stationary phase to accomplish particular separations. This entails how well the column is packed and its kinetic performance (Bidlingmeyer, 1984). The efficiency of a column can be measured by several methods which may or may not be affected by chromatographic anomalies, such as "tailing" or appearance of a "front." This is important because many chromatographic peaks do not appear in the preferred shape of normal Gaussian distribution. For this reason efficiency can be an enigmatic value since manufacturers may use different methods in determining the efficiency of their columns (Bidlingmeyer, 1984).

Calculation of column efficiency value:

All the following methods use this formula that measures N, or number of theoretical plates:
.

Inflection Method- Calculation is based upon inflection point which appears at 60.7% of the peak height for a normal Gaussian peak. At this point the width of the peak is equivalent to two standard deviation units. Any asymmetrical aspect of a peak should not affect this calculation since the width is measured above the anomalous occurance (i.e., tailing or fronting).
(Bidlingmeyer, 1984)

Half-peak height Method- As the name suggests, the measurement is based upon the width at 50% of peak height. For the same reason as inflection method, this measurement is not affected by asymmetry; however, this method is more reproducible from person to person since width at 50% peak height is less prone to be varied.
(Bidlingmeyer, 1984)

Tangent Method- Tangent lines are drawn on each side of the peak and the width is the distance between the two lines at the base of the peak. Therefore, it is more sensitive to asymmetrical peaks and variation in efficiency values is usually seen from user to user.
(Bidlingmeyer, 1984)

Sigma Methods- These methods measure peak width at decreasing levels of peak height. Thus, the three sigma method measures width at 32.4% of peak height, the four sigma method measures at 13.4%, and the five sigma method measures at 4.4%. The five sigma method is most sensitive to asymmetry because the width is measured at the lowest point.
(Bidlingmeyer, 1984)

Height/Area Method- This method utilizes the fact that the area of a peak is a function of its height and standard deviation. To determine efficiency, values for peak height and area are used in a different formula:
A computer is usually necessary to use this method in order to calculate the area and height.

Moment Method- This method entails disregarding peak shape and expresses parameters of the peak in statistical moments. The zero moment, µ0, is the peak area. The first moment, µ1, is the mean and occurs at the center of the peak (which is the maximum peak height in normal Gaussian peaks). The second moment, µ2, is the variance of the peak. This is a detailed method where appropriate data systems are needed. For a more detailed discussion, a reference is provided (Grubner, 1958).

These methods were evaluated by computer simulation based on efficiency values obtained on a series of synthetically modified Gaussian peaks (i.e., increasing the 'tailing') and compared to the actual value based on the moment method (which was determined to be the most accurate). Briefly, the results were as follows:

CALCULATION METHOD--ACCURACY(Bidlingmeyer, 1984).
Inflection Low
Half-peak height Low
Tangent Low
Height:Area ratio Medium
Four sigma Medium
Five sigma High
Asymmetry High
Bidlingmeyer, B.A. and Warren, F.V. Jr. Analytical Chemistry, 1984, Vol. 56, pp. 1583-96.Grubner, O. Advances in Chromatography, Giddings, J.C. and Keller, R.A. Eds.; Marcel Dekker: New York, N.Y., 1958, Vol. 6, pp. 173-209.