Header Ads Widget


SOP for Verification of HPLC Column Performance

This SOP provides a guide on how to verify the performance of the HPLC column. The verification of column performance is carried out in order to assess whether the specifications determined by the vendor are met when the column is first used in the laboratory. Moreover, when the column is routinely used, its performance will decline hence it is important to monitor its performance over time using the same verification procedure. When the column is new, the test is performed using all of the HPLC instruments found in the laboratory in order to have a reference for future verification.

This procedure is applicable to the verification of a new column as well as to routinely used columns whose performance needs to be verified.


Plate number (N)
A number indicative of column performance, calculated from the following equation:
tr: for the retention time 
wh: for the peak width at half height

USP Tailing factor (Tf)
Used to measure the degree of peak symmetry and is calculated using the following equation:
W0.05: Peak width at 5% height 
f: Front half-width at 5% of the peak height.

Capacity factor or Retention Factor (k´)
A measure of the time the sample component resides in the stationary phase relative to the time it resides in the mobile phase; it expresses how much longer a sample component is retarded by the stationary phase than it would take to travel through the column with the velocity of the mobile phase. It is calculated as
tr: for the retention time 
t0: for the retention time of the unretained compound

Selectivity (∝)
The relative retention value was calculated for two adjacent peaks. It is also called the separation factor and is calculated as

QC Analyst: is responsible for verifying column performance 
QC Manager: ensuring that the column performance is monitored.

Evaluate the column efficiency using the chromatographic test conditions and test compounds indicated on the HPLC column performance result. Perform the test on a new column to verify its performance. Repeat the test periodically to monitor the column performance.
  • Prepare a solution of the test compounds necessary for the column test. The test compounds are: 20 μg/mL uracil, 30 μg/mL acetophenone, μg/mL 4-Cl-nitrobenzene, and 2.40 mg/mL toluene in 50:50 (v/v) acetonitrile: water. It is necessary that these 4 test compounds are available. However, in case one of the test compounds (acetophenone or 4-Cl-nitrobenzene) is not available, the test must still be performed with the 3 compounds comprising the test solution.
  1. 50:50 (v/v) acetonitrile: water solution. Measure 25 mL of HPLC-MS grade acetonitrile and mix with 25 mL of HPLC-MS grade water.
  2. 1 mg/mL uracil solution. Weigh 5 mg of uracil and dissolve in 5 mL of 50:50 (v/v) acetonitrile: water solution.
  3. 1 mg/mL acetophenone solution. Pipet 5μL of acetophenone and mix with 5 mL of 50:50 (v/v) acetonitrile: water solution.
  4. 1 mg/mL 4-Cl-nitrobenzene solution. Weigh 5 mg of 4-Cl-nitrobenzene and dissolve in 5 mL of 50:50 (v/v) acetonitrile:water solution.
  5. 10 mg/mL toluene solution. Pipet 58 μL of toluene and mix with 5 mL of 50:50 (v/v) acetonitrile: water solution
  6. Column test solution. Pipet 20 μL of 1 mg/mL uracil solution, 30 μL of 1 mg/mL of acetophenone solution, 77 μL of 1 mg/mL 4-Cl-nitrobenzene solution, 240 μL of 10 mg/mL toluene solution and 367 μL of 50:50 (v/v) acetonitrile: water solution into a glass vial and mix.

Prepare the HPLC instrument for analysis.
  • Fill the respective mobile phase reservoirs with water and acetonitrile.
  • Turn on the HPLC modules and the computer connected to the HPLC instrument. In the computer, create a file name for the column test. Create a method for performing the column test. The chromatographic conditions are as follows.
  1. Mobile phase: 50:50 acetonitrile: water
  2. Flow rate: 0.5 mL/min
  3. Injection volume: 1 μL
  4. Acquisition time: 4 min
  5. Temperature: 25°C
  • Purge the HPLC solvent lines.
  • Connect the HPLC column.
  • Place the vial of the test solution in the autosampler rack.
  • Equilibrate the column with the mobile phase for at least 15 minutes before injecting the test compound.
  • Obtain the test chromatogram and determine the number of theoretical plates (N), USP tailing factor (Tf), selectivity and capacity factor (k´) for the toluene peak. The calculations can be done manually or by HPLC software.
  • Compare the results with the vendor´s test chromatogram specification. To assess whether the column passed the performance test, the following must be checked:
  1. the test compounds must appear in the chromatogram
  2. the retention times are not significantly different
  3. the values for the number of plates, tailing factor, selectivity, and capacity factor are within the minimum specifications set by the vendor.
  • If the column is new, repeat the test using the other HPLC instrument found in the laboratory.

  • If the determined N value for a new column is < 80% of the specified value, determine first whether there is a possibility of instrumental problems and make the necessary corrections. Repeat the test using the same conditions.
  • For already used columns, the results for the N value will be normally lower than the previous determinations. If the N value decreases by 50% then the column may be replaced.


Revision History

Post a Comment