HPLC: Tutorial

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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

  1. column dimension (size)
  2. particle size and pore size
  3. stationary phase
  1. 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.
  2. 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.

The stationary phase is generally made up of  hydrophobic alkyl chains ( -CH2-CH2-CH2-CH3 ) 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.

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