Figure 1. Influence of sample solvent on peak shape. The sample is dissolved in buffer with (a) 0%, (b) 30%, (c) 50%, and (d) 70% acetonitrile. Sample Injection (Mobile Phase 92% buffer pH 3.5, 8% acetonitrile)

Column Testing The best way to evaluate a column’s performance is to use the same test that was used by the manufacturer before it was shipped. By comparing the efficiency, retention and peak shape of the peaks in the sample and very importantly the pressure under these experimental conditions, you will be able to tell if your column has changed over time.

Column Regeneration Cleaning Reversed-Phase Columns Use 10 column volumes of each: 100% methanol 100% acetonitrile 75% acetonitrile/25% isopropanol 100% methylene chloride 100% hexane 100% isopropanol Aqueous based solvents such as acetic acid, trifluoroacetic acid, urea, guanidine, sodium phosphate, or dimethylformamide can aid in the removal of proteinaceous material from reversed phase columns.

Gradient Testing Mobile Phase A = Methanol Mobile Phase B = 1000 mL methanol with 2 mL acetone Flow = 2 mL/min Detector 280nm The acetone in Mobile phase B is detectable by UV at 280 nm. Therefore, as the amount of Mobile Phase B is increased, the detector response increases. GRADIENT 0 min97.5% A and 2.5 % B 10 min 2.5% A and 97.5% B 12 min 2.5% A and 97.5% B 12.1 min97.5% A and 2.5 % B 20 min97.5% A and 2.5 % B

Evaluation of the Gradient Test. Gradient Testing Repeat 4-5 Times and note standard deviations of times and detector response.

Gradient Testing

Should be linear Not too much curvature Not too much dwell time

Dwell Time Dwell Time is the time it takes the mobile phase to move through the dwell volume. The dwell volume is the volume of the mobile phase between where the solvents are mixed and the column. This includes the mixing chamber, the injector, and tubing. Depending on the instrument, dwell volumes can be as low as 0.5 mL or as high as 8.0 mL. Differences in dwell volume can cause considerable differences in chromatography and may be one reason that it is difficult to reproduce a separation reported in the literature. In documenting a method, the dwell volume of the instrument should be reported.

Extra-Column Volume Extra-column volume is volume of the system other than the column that contributes to band broadening outside the column, (in addition to the dwell volume, the detector flow cell, post column reactors, and tubing are the main sources). The relative amount of allowable extra column broadening is proportional to the column volume. The relative volumes of 2 columns can be calculated based on the formula for the volume of a cylinder. Length x  R 2. Example: (Column 1, diameter = 4.6 mm, extra column volume = 100  L, Column 2, diameter = 2 mm, What is the equivalent extra-column volume?) Since the radius(R) is the only thing changing in this example, relative volume can be calculated as the ratio of the two radii ( /1 2 = 5.3) which means that the 2 mm column has a volume 5.3 times less than a 4.6 mm column therefore, the extra-column volume should be 5.3 times less (100  L/5.3 = 18  L).

Problem 1. No Peaks/Very Small Peaks

Problem 2. No Flow

Problem 3. No Pressure or Pressure Lower than Usual

Problem 4. Pressure Higher than Usual

Problem 5. Variable Retention Times

Problem 6. Loss of Resolution

Problem 7. Split Peaks

Problem 8. Peaks Tail on Initial and Later Injections

Problem 9. Tailing Peaks

Problem 10. Fronting Peaks

Problem 11. Rounded Peaks

Problem 12. Baseline Drift

Problem 13. Baseline Noise (Regular)

Problem 14. Baseline Noise (Irregular)

Problem 15. Broad Peaks

Problem 16. Change in Peak Height (one or more peaks)

Problem 17. Change in Selectivity

Problem 18. Negative Peaks

Problem 19. Ghost Peaks