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% methanol100% acetonitrile75% acetonitrile/25% isopropanol100% methylene chloride100% hexane100% isopropanol

Aqueous based solvents such as acetic acid, trifluoroaceticacid, urea, guanidine, sodium phosphate, ordimethylformamide can aid in the removal of proteinaceousmaterial from reversed phase columns.

Gradient Testing

Mobile Phase A = MethanolMobile Phase B = 1000 mL methanol with 2 mL acetoneFlow = 2 mL/minDetector UV @ 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 min 97.5% A and 2.5 % B10 min 2.5% A and 97.5% B12 min 2.5% A and 97.5% B12.1 min 97.5% A and 2.5 % B20 min 97.5% A and 2.5 % B

Evaluation of the Gradient Test.

Gradient TestingRepeat 4-5 Times and note standard deviations oftimes and detector response.

Gradient Testing

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 movethrough the dwell volume. The dwell volume is the volume of the mobile phase between where the solvents aremixed 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 considerabledifferences in chromatography and may be one reason thatit is difficult to reproduce a separation reported in theliterature. In documenting a method, the dwell volume ofthe instrument should be reported.

Extra-Column VolumeExtra-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 proportionalto the column volume. The relative volumes of 2 columns can be calculated based on the formula for the volume of a cylinder. Length x R2.

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 ( 2.32/12= 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