High Performance Liquid Chromatography

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

Instrument/Software Operation

1) Turn on all instrument components using the single 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.

Where to go from here? If you are reading this page, then you are probably new to the instrument and will be starting at the beginning:

Select/define an HPLC "Method"

1) If not done so previously, please read over the "Method Development" section below.

2) You will need to decide on the following:

- mobile phases conditions...isocratic or gradient?

if isocratic, then mobile phase percentages

if gradient, then mobile phase gradient profile/conditions

- Most appropriate UV wavelength to monitor analyte

3) once method is defined, equilibrate to column with this method (ie. starting conditions) and then move on to loading samples.

Autosampler: Load samples into Tray

1) Autosample tray is accessed by manually opening to door by pulling from the top.

2) the tray will disengage after ~10 seconds

3) Load sample vials making note of the location in the tray

4) re-insert tray into autosampler and close door; tray will automatically re-engage.

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

Previous Analysis

Caffeine_HPLC_Method Hop_Isomerization