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Reverse Phase HPLC Basics for LC/MS

An IonSource Tutorial

by

Andrew Guzzetta

This Tutorial was first published July 22nd, 2001

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We were going to call this tutorial "Reverse Phase HPLC for Proteomics" but we decided to exercise
some restraint. We decided to write this tutorial because reverse phase chromatography is the most
common form of chromatography used in LC/MS applications. This tutorial is basically targeted to
students and those that are new to reverse phase chromatography, HPLC, and LC/MS. The tutorial
addresses RP HPLC of peptides and proteins but the principles described can be applied toward the
chromatography of any compound.

Table of Contents
 1. Introduction

2. The HPLC

3. The Column

4. Solvents

5. Gradient

6. Flow Rate

7. Sample Preparation

8. Optimizing the Separation

9. What is HPLC Equilibration?

10. Preparing for the First Run of the Day

11. After the Last Run of the Day

12. Should I Control Column Temperature?

13. How Much Protein Should I Load?

14. Links to Related IonSource Material

15. Links to External HPLC Information

Introduction

The message of this tutorial is that reverse phase HPLC is simple. Compounds stick to reverse phase
HPLC columns in high aqueous mobile phase and are eluted from RP HPLC columns with high
organic mobile phase. In RP HPLC compounds are separated based on their hydrophobic character.
Peptides can be separated by running a linear gradient of the organic solvent. I often tell my fellow
researchers to run the 60/60 gradient when chromatographing an unknown. The 60/60 gradient
means that the gradient starts at near 100% aqueous and ramps to 60% organic solvent in 60
minutes. The majority of peptides (10 to 30 amino acid residues in length) will elute by the time the
gradient reaches 30% organic. To learn some of the simple principles of RP HPLC please read on.
The HPLC

In most cases the HPLC you intend to use must be able to pump and mix two solvents. This can be
accomplished with one pump and a proportioning valve or by using two separate pumps. Generally
the pumping configuration is an aspect of the instrumentation that is transparent to the user. Reverse
phase chromatography can also be performed in a purely isocratic mode where the solvent conditions
are held constant, this form of reverse phase chromatography can be carried out with a single pump.
Isocratic methods are used most often in a QC environment in which a single analyte has been
extensively characterized and the compound is being run to confirm it's identity and to look for
closely related degradation products. If you do not own an HPLC here is a link to HPLC vendors
and accessory suppliers.

HPLC Column Components and Specifications

a. column dimension (size)

b. particle size and pore size

c. stationary phase

a. Since columns are tubular, column dimensions usually take the following format,
internal diameter X length (4.6mm X 250mm). As a mass spectroscopist you will
encounter columns ranging in internal diameter from 0.050 to 4.6 mm or even larger
if you are performing large scale preparative chromatography. For mass spectrometry
a short reverse phase column will work nearly as well as a longer column and this is
an important fact because shorter columns are generally cheaper and generate less
back pressure. Why is less back pressure important? If a column runs at low pressure
it allows the user more flexibility to adjust the flow rate. Sometimes shorter columns
are used to do fast chromatography at higher than normal flow rates. In terms of
length we routinely run 100 mm columns, however 50 mm or 30 mm columns may be
adequate for many LC/MS separation needs.

b. The most common columns are packed with silica particles. The beads or particles
are generally characterized by particle and pore size. Particle sizes generally range
between 3 and 50 microns, with 5 um particles being the most popular for peptides.
Larger particles will generate less system pressure and smaller particles will generate