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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 µm particles being the most popular for peptides. Larger particles
will generate less system pressure and smaller particles will generate more pressure. The
smaller particles generally give higher separation efficiencies. The particle pore size is
measured in angstroms and generally range between 100-1000 angstroms. 300 angstroms is
the most popular pore size for proteins and peptides and 100 angstroms is the most common
for small molecules. Silica is the most common particle material. Since silica dissolves at
high pH it is not recommended to use solvents that exceed pH 7. However, recently some
manufactures have introduced silica based technology that is more resistant to high pH, it is
important to take note of the manufactures suggested use recommendations. In addition the
combination of high temperature and extremes of pH can be especially damaging to silica.
c.
The stationary phase is generally made up of hydrophobic alkyl chains ( -CH2-CH2-CH2CH3 ) that interact with the analyte. There are three common chain lengths, C4, C8, and
C18. C4 is generally used for proteins and C18 is generally used to capture peptides or small
molecules. The idea here is that the larger protein molecule will likely have more
hydrophobic moieties to interact with the column and thus a shorter chain length is more
appropriate. Peptides are smaller and need the more hydrophobic longer chain lengths to be
captured, so C8 and C18 are used for peptides or small molecules. Here is an interesting
note: Observations have been made that C8 columns are actually better for capturing smaller
hydrophilic peptides, the theory here is that the longer C18 chains lay down during the early
aqueous period of the gradient and the more hydrophilic peptides are not captured. We use
C8 routinely for all peptide work and this particular alkyl chain length works equally well if
not better than C18 for all peptides.
Solvents
The reverse phase solvents are by convention installed on the HPLC channels A and B. The A
solvent by convention is the aqueous solvent (water) and the B solvent by convention is the organic
solvent (acetonitrile, methanol, propanol). It is important to follow this convention since the terms
A and B are commonly used to refer to the aqueous and organic solvents respectively. The A solvent
is generally HPLC grade water with 0.1% acid. The B solvent is generally an HPLC grade organic
solvent such as acetonitrile or methanol with 0.1% acid. The acid is used to the improve the
chromatographic peak shape and to provide a source of protons in reverse phase LC/MS. The acids
most commonly used are formic acid, triflouroacetic acid, and acetic acid. A 0.1% v/v solution is
made by adding 1ml of acid per liter of solvent. Triflouroacetic acid has been reported to suppress
MS ionization and often mass spectroscopists lower the percentage of TFA to 0.05 or even 0.02%
without significant loss in chromatographic efficiency. Some MS people add a small percentage of
heptafluorobutyric acid (HFBA, pdf from Michrom) to acetic acid solvents or low TFA containing
solvents to help improve peak shape. Since modern mass spectrometers are very sensitive it is
important not to use plastic pipette tips when adding acid to the mobile phase, always use glass. In
our work we use acetonitrile as our organic solvent. We have heard that the best electrospray solvent
is 30% methanol, 35 mM acetic acid. We commonly use this solvent system for ESI MS infusion,
but have found that acetic acid is an inferior acid for chromatographic peak shape. Our preferred
HPLC grade water, acetonitrile and methanol is purchased from Burdick and Jackson. Our preferred
TFA comes in 1 ml ampoules from from Pierce Chemical Company.
Our Preferred Solvent System for ESI LC/MS
A = HPLC grade Water, 0.1 % formic acid
B = HPLC grade Acetonitrile, 0.1% formic acid
Gradient
When chromatographing an unknown we normally use the following simple gradient to learn about
the hydrophobic character of the unknown compound. The % A in the gradient described below is
implied.
We call this the 60/60 gradient, because we run from near 0% B to 60% B in 60 minutes. Through
experience we have noted that 90% of all peptides will elute from a C18 reverse phase column by
30% acetonitrile. There may be a few really hydrophobic peptides that elute later that is why we take
the gradient to 60% B. You may even want to run this gradient to 80% at least once to see if you are
getting everything off of the column. You may ask why don't we start the gradient at 0% B? As we
talked about before, in 0% organic and in high aqueous, the very hydrophobic, long C18 alkyl chains
in an effort to get away from the high aqueous environment mat down on the particle. When these
alkyl chains mat down they are inefficient at capturing the analyte so chromatographers in the know
start the gradient with some small % of organic, 2-5%.
Flow Rate
It is important to use the correct flow rate for your HPLC column. Below is a table with standard
flow rates for easy reference. If you are running a column with a different diameter than those
shown in the table please review the maintaining linear velocity page to learn how to calculate the
appropriate flow rate for your column.
Sample Preparation
The sample is normally reconstituted in the A solvent to maximize binding to the column. The
sample should not be dissolved in an organic solvent or it may not stick to the stationary phase. The
sample should not be dissolved in detergent containing solutions. Some detergents may bind to
reverse phase columns and modify them irreversibly. In addition detergents preferentially ionize in
electrospray mass spectrometry and can obscure the detection or suppress the ionization of the
analyte.
Optimizing the Separation
Once you have a separation you may want to optimize it. You may wish to shallow out the gradient
to improve the separation, or you may wish to shorten the run time. Taking the illustration above
one can see that all of the peptides are out by 40 minutes. This does not mean that we can change
this 80 min run into a 40 min run, but there is room for improvement. The first step in the
optimization is to determine the %B at which the last peak elutes. If you look at the blue gradient
line you might guess that the last peak elutes near 40%B but this would be incorrect. All HPLC
systems have a gradient delay. The gradient delay is the time between when the software tells the
pumps to start pumping at a certain mobile phase composition and the time it takes for that solvent
composition to reach the column and have an effect. A good guess for a gradient delay is 10
minutes. This would mean that our guess for the final mobile phase composition for the 40 min peak
would be approximately 30%B. To observe the gradient delay time look at the illustration above and
observe that the baseline returns to the starting conditions at 70 minutes and not at 60.1 minutes
when our pumps have gone back to 2% B. One must take care to avoid having the last peak elute on
the "equilibration cliff", (at 70 min. in this example). This can be avoided by ending the gradient at a
%B that is slightly higher than that required by the last component.
Based on the separation shown at the top of this section one could rewrite the gradient to look like
this:
This would make the gradient shallower and possibly give a better peak separation. To shorten the
run time one could rewrite the gradient to look like this:
This last change would cut 30 min. from the analysis time. Shorter analysis times are always better
for work efficiency. With every minute you can cut from the HPLC method without sacrificing your
chromatographic goals you will be rewarded with better work efficiency. With this change the last
peak would most likely still elute at 40 minutes and the peptide separation would essentially remain
the same as in the initial 60/60 analysis.
What is HPLC Equilibration?
The column must be equilibrated, re-equilibrated to the initial high aqueous solvent composition
before another analysis can be performed. Normally this re-equilibration is stuck onto the end of the
gradient. How much equilibration time is enough? As a rule of thumb we give 20 minutes. In
reality it depends on the column length, flow rate and the hydrophobicity of your peptides. Some
chromatographers use 10 minutes as their standard equilibration time. Equilibration is all about
fitness of purpose. You should determine the the equilibration time experimentally, the criteria will
be, does my analyte really stick to the column and chromatograph appropriately and reproducibly
with subsequent analyses. If you choose to do this part of the method development you will
undoubtedly be rewarded with improved chromatography and better cycle time.
Should I Control Column Temperature?
Yes. Scientists are control freaks. If you can control a variable, control it! Actually if you are
performing automated analyses over a long period of time peak retention times can drift with
changing ambient temperature. It is common for many companies and institutions shut down the air
conditioning at night to save money, which could result in shifting peak retention times due to
dramatic changes in ambient temperature. Many HPLCs provide the option to control column
compartment temperature. If your HPLC does not have this capability a heated column jacket can be
purchased from many suppliers. The most common running temperature is 40°C, this places the
column compartment well above even the most extreme ambient temperature fluctuations. In
addition to maintaining constant temperature, temperature can be used to influence the
chromatographic separation. No chromatographic study is complete without a temperature study. In
our experience higher temperature is better, peaks will be sharper and elute earlier. It is not too
uncommon to perform chromatography at 60°C and some daredevils even go to 80°C. Remember
though that higher temperature will lead to a shorter column lifetime and some columns may not be
able to tolerate 60°C. Consult the manufactures recommendations when experimenting with high
temperature. After your runs are complete for the day it is advised that you turn off your column
heater since high temperature leads to stationary phase deterioration.
Preparing for the First Run of the Day
One observation is that if you start up a reverse phase analysis from a dead stop with a column that
has perhaps been sitting in high aqueous conditions for up to 10 hours the analysis will give
irreproducible results. Conventional wisdom has it, you want to first flush the column with the
highest % organic of your method for at least 3 column volumes and then bring it back to the
equilibrating condition. This practice may have the advantage of getting you to standard
equilibration conditions faster and it will also clean your column. A better alternative is to make the
first run a blank run (or "preparation run") and then the next run can be your real analysis. We prefer
the second option because it should get you to the standard starting conditions more accurately.
However, often, if we are in a hurry and the first option is quicker, well.....
After the Last Run of the Day
We store our columns in 50/50 methanol/water without any acid. If you are using a salt, unlikely in
LC/MS, wash your entire system, solvent bottles, HPLC, solvent lines, and column, into a non-salt
containing solvent. Salt may precipitate out and plug your HPLC or column or may cause
corrosion. Usually we flush with pure water first then leave the system in 50/50 methanol: water.
Some salts may precipitate out in high organic so an initial water wash is advised. The 50/50
methanol:water solution helps to stop bacterial growth which can muck up your system. Take care
of your HPLC, it's the right thing to do!
How Much Protein Should I Load?
Links to Related IonSource Material
Calculating the Appropriate Flow Rates for Columns of Differing Diameters, Maintaining Linear
Velocity
HPLC Tubing Volume Calculator
Make an HPLC Sample Injection Loop
Capillary HPLC Tutorial
HPLC and Accessory Vendors
Links to External HPLC Information
Basic Liquid Chromatography
Vydac's, "The Handbook of Analysis and Purification of Peptides and Proteins by Reversed-Phase
HPLC"
HPLC Trouble Shooter
HPLC Column Volume
Useful HPLC Tables & Charts
Chromatography at Yahoo
Quality HPLC Links from LCResources
Important Safety Information: Triflouroacetic acid, formic acid, heptaflouobutyric acid and acetic acid are all very
caustic reagents. Acetonitrile, methanol, and propanol are harmful solvents Consult the material safety data sheets
(MSDS) that come with these reagents and get the permission of the safety officer at you company or institution before
performing these experiments. Always wear the appropriate safety apparel; safety glasses, lab coat, and gloves. Use a
fume hood when appropriate. If you are not trained in laboratory safety you should not attempt these procedures. Read
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Calculating the Appropriate Flow Rates for Columns of
Differing Diameters
or
"Maintaining Linear Velocity"
An IonSource.Com mini-tutorial
by
Andrew Guzzetta
Introduction
(flow rate, pressure and chromatography)
The standard size HPLC column (4.6 X 250 mm) is run at a flow rate of 1 mL/min. 4.6 refers to the
internal diameter and 250 refers to the length of the HPLC column in millimeters. The limiting factors in
choosing a flow rate are, instrument pressure limitations, the effect on the quality of the chromatography,
and time. Most HPLC's operate in the pressure range between 30 and 200 bar. Maintaining linear
velocity is the single most important factor when trying to reproduce a chromatographic separation on
columns of differing diameters. Since flow rate directly impacts HPLC system pressure a discussion of
parameters that affect system pressure are listed below.
Factors that affect system pressure:
Column Dimension
Shorter, fatter columns produce lower system pressures, which allows them to be run at higher flow
rates. Often the chromatographer will exploit this and run short fat columns at higher flow rates to speed
up analysis times. HPLC peaks do not always get narrower with increased flow rate. At faster flow rates
the analyte may have insufficient time to interact with the stationary phase.
Stationary Phase Particle Size
Stationary phase particles range in size from 2 to 25 microns. The most common particle size is 5
micron. In general the larger the particle the lower the system pressure for a given flow rate. Smaller
particles generally provide greater surface area and better separation but produce higher system pressures.
Temperature
System pressure is affected by temperature. The viscosity of the mobile phase will decrease with
increasing temperature. For example if the HPLC system pressure is too high for a given solvent system
the chromatographer may choose to raise the temperature of the column compartment to 40°C or even
60°C. In terms of trouble shooting high system pressure, adjusting temperature in not usually the first
factor to consider. Often an increase in back pressure will be a sign of column plugging or fouling.
Flow Rate
It is not difficult to imagine that system pressure is affected by flow rate. Flow rate impacts HPLC
system pressure, chromatographic quality, and analysis time. One must choose a flow rate that is
appropriate for the HPLC system and column. A higher than usual flow rate may adversely affect the
quality of the chromatography not giving the analyte sufficient time to interact with the stationary phase.
Faster isn't always better. A lower than usual flow rate may leave the analyst waiting for the peak to
appear at the detector.
"Maintaining Linear Velocity"
.
Maintaining mobile phase linear velocity is important when attempting to reproduce chromatography
obtained on columns of differing internal diameters. For example to maintain mobile phase linear
velocity from the standard column mentioned above, 4.6 X 250 mm, to a small bore column with the
dimensions 2.1 X 250 mm one must adjust the flow rate downward by a factor. When going from a 4.6
to a 2.1mm ID or to any smaller ID column decrease the flow rate by the square of the ratios of the
column diameters. As mentioned above the standard flow rate for a 4.6 mm column is 1 ml/min.
In our example the new flow rate determined for the 2.1 mm column is 0.208 ml/min. Commonly 2.1
mm columns are run at a flow rate of 200 µl/min.
It is important to note that sometimes adjustments in mobile phase flow rates from the standard flow rate
can have minor beneficial effects in chromatography. Sometimes flowing a column at one half the
normal flow rate will have a beneficial effect on a chromatographic separation.
.
Good Luck.
Happy Chromatography.
Sincerely,
Andrew Guzzetta
.
.
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