High Performance Liquid Chromatography

The following page is related to the Waters Breeze HPLC system.

Instrument Operation

1) Turn on all instrument components using the powerstrip "ON" switch.

2) Wait 1 min and then launch the "Breeze" software.

- depending on the previous state of the system, the boot time varies; do nothing until the "flow monitoring" status window appears.

3)

Method Development

This HPLC currently has a C18 column and is setup to conduct "reversed phase" HPLC analysis. This means that the stationary phase (the column packing material) is non-polar and that the mobile phase under "isocratic conditions" is more polar than the stationary phase. When samples are analyzed using a mobile phase "gradient," the mobiles phase transitions from being highly polar (ie. water) to less polar (ie. acetonitrile). Any solvents used during a gradient MUST be miscible. Typical mobile phases in a reversed phase HPLC analysis are:

1) acidified (0.1% TFA) H₂O and acetonitrile

2) 50% Methanol/?% phosphoric acid and Methanol

(Note: acidified water and methanol cannot be used due to the heat of dissolution and the resulting degassing of the solvents)

Sample Preparation

HPLC is an analytical technique that analyzes very dilute samples. The term "sample load" is often used to make sure that samples are suitable for HPLC analysis.

Caffeine example

- consider a caffeine stand made by dissolving 17 mg of caffeine in 100 mL of RO water.

- a traditional 10 ul injection of this caffeine standard will place ~2 ug of caffeine on the HPLC column.

- the lambda max absorption (273 nm) of the eluting caffeine is ~ 1 absorbance unit.

- the "sample load" in this example is ~2 ug and appears to be a reasonable target value.

(Note: sample loads can be significantly lower than 2 ug and still be able to detect the analyte)

Before injecting into the HPLC...

1) do you anticipate the sample loading is ~2 ug or less? If not, evaluate this using UV-Vis and the molar absorbance of your analyte.

(if this sample is an unknown, then inject a highly diluted sample first.)

2) Unless samples are made from highly purified standards, all samples need to be filtered using a 0.45 um syringe filter.

3) Is there sufficient mobile phase to analyze all of your samples (the HPLC system does not monitor mobile phase levels).

Consumables

- sample vials/caps

- 0.45 um syringe filters for aqueous (###) and organic (XXX) samples

- HPLC column

- HPLC guard column inserts