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Reverse Phase HPLC Basics for LC/MS

An IonSource Tutorial


Andrew Guzzetta

This Tutorial was first publishedJuly 22nd, 2001
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read important laboratory safety notice at bottom of page before proceeding

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

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.


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 um 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.
  3. 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.

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

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%.

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.

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.

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.

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.

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 40C, 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.

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...

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!

Links to Related IonSource Material
Calculating the Appropriate Flow Rates for Columns of Differing Diameters, Maintaining Linear Velocity

HPLC and Accessory Vendors

Links to External HPLC Information
ImportantSafety 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 our disclaimer, follow the link at the bottom of this page.

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Last updated: Tuesday, January 19, 2016 02:49:16 PM


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It’s all about data these days. Leaders don’t want to make decisions unless they have evidence. That’s a good thing, of course, and fortunately there are lots of ways to get information without having to rely on one’s instincts. One of the most common methods, particularly in online settings, is A/B testing.

To better understand what A/B testing is, where it originated, and how to use it, I spoke with Kaiser Fung, who founded the applied analytics program at Columbia University and is author of Junk Charts, a blog devoted to the critical examination of data and graphics in the mass media. His latest book is Number Sense: How to Use Big Data to Your Advantage.

What Is A/B Testing?

A/B testing, at its most basic, is a way to compare two versions of something to figure out which performs better. While it’s most often associated with websites and apps, Fung says the method is almost 100 years old.

In the 1920s statistician and biologist Ronald Fisher discovered the most important principles behind A/B testing and randomized controlled experiments in general. “He wasn’t the first to run an experiment like this, but he was the first to figure out the basic principles and mathematics and make them a science,” Fung says.

Fisher ran agricultural experiments, asking questions such as, What happens if I put more fertilizer on this land? The principles persisted and in the early 1950s scientists started running clinical trials in medicine. In the 1960s and 1970s the concept was adapted by marketers to evaluate direct response campaigns (e.g., would a postcard or a letter to target customers result in more sales?).

A/B testing, in its current form, came into existence in the 1990s. Fung says that throughout the past century the math behind the tests hasn’t changed. “It’s the same core concepts, but now you’re doing it online, in a real-time environment, and on a different scale in terms of number of participants and number of experiments.”

How Does A/B Testing Work?

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You start an A/B test by deciding what it is you want to test. Fung gives a simple example: the size of the subscribe button on your website. Then you need to know how you want to evaluate its performance. In this case, let’s say your metric is the number of visitors who click on the button. To run the test, you show two sets of users (assigned at random when they visit the site) the different versions (where the only thing different is the size of the button) and determine which influenced your success metric the most. In this case, which button size caused more visitors to click?

In real life there are lots of things that influence whether someone clicks. For example, it may be that those on a mobile device are more likely to click on a certain size button, while those on desktop are drawn to a different size. This is where randomization can help — and is critical. By randomizing which users are in which group, you minimize the chances that other factors, like mobile versus desktop, will drive your results on average.

“The A/B test can be considered the most basic kind of randomized controlled experiment,” Fung says. “In its simplest form, there are two treatments and one acts as the control for the other.” As with all randomized controlled experiments, you must estimate the sample size you need to achieve a statistical significance, which will help you make sure the result you’re seeing “isn’t just because of background noise,” Fung says.

Sometimes, you know that certain variables, usually those that are not easily manipulated, have a strong effect on the success metric. For example, maybe mobile users of your website tend to click less on anything, compared with desktop users. Randomization may result in set A containing slightly more mobile users than set B, which may cause set A to have a lower click rate regardless of the button size they’re seeing. To level the playing field, the test analyst should first divide the users by mobile and desktop and then randomly assign them to each version. This is called blocking.

The size of the subscribe button is a very basic example, Fung says. In actuality, you might not be testing just the size but also the color, and the text, and the typeface, and the font size. Lots of managers run sequential tests — e.g., testing size first (large versus small), then testing color (blue versus red), then testing typeface (Times versus Arial) — because they believe they shouldn’t vary two or more factors at the same time. But according to Fung, that view has been debunked by statisticians. And sequential tests are suboptimal because you’re not measuring what happens when factors interact. For example, it may be that users prefer blue on average but prefer red when it’s combined with Arial. This kind of result is regularly missed in sequential A/B testing because the typeface test is run on blue buttons that have “won” the prior test.

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Instead, Fung says, you should run more-complex tests. This can be hard for some managers, since the appeal of A/B tests are how straightforward and simple they are to run (and many people designing these experiments, Fung points out, don’t have a statistics background). “With A/B testing, we tend to want to run a large number of simultaneous, independent tests,” he says, in large part because the mind reels at the number of possible combinations you can test. But using mathematics you can “smartly pick and run only certain subsets of those treatments; then you can infer the rest from the data.” This is called “multivariate” testing in the A/B testing world and often means you end up doing an A/B/C test or even an A/B/C/D test. In the example above with colors and size, it might mean showing different groups: a large red button, a small red button, a large blue button, and a small blue button. If you wanted to test fonts, too, the number of test groups would grow even more.

How Do You Interpret the Results of an A/B Test?

Chances are that your company will use software that handles the calculations, and it may even employ a statistician who can interpret those results for you. But it’s helpful to have a basic understanding of how to make sense of the output and decide whether to move forward with the test variation (the new button in the example above).

Fung says that most software programs report two conversion rates for A/B testing: one for users who saw the control version, and the other for users who saw the test version. “The conversion rate may measure clicks, or other actions taken by users,” he says. The report might look like this: “Control: 15% (+/- 2.1%) Variation 18% (+/- 2.3%).” This means that 18% of your users clicked through on the new variation (perhaps your larger blue button) with a margin of error of 2.3%. You might be tempted to interpret this as the actual conversion rate falling between 15.7% and 20.3%, but that wouldn’t be technically correct. “The real interpretation is that if you ran your A/B test multiple times, 95% of the ranges will capture the true conversion rate — in other words, the conversion rate falls outside the margin of error 5% of the time (or whatever level of statistical significance you’ve set),” Fung explains.

If this is hard to wrap your head around, join the club. What’s important to know is that the 18% conversion rate isn’t a guarantee. This is where your judgment comes in. An 18% conversation rate is certainly better than a 15% one, even allowing for the margin of error (12.9%–17.1% versus 15.7%–20.3%). You might hear people talk about this as a “3% lift” (lift is simply the percentage difference in conversion rate between your control version and a successful test treatment). In this case, it’s most likely a good decision to switch to your new version, but that will depend on the costs of implementing the new version. If they’re low, you might try out the switch and see what happens in actuality (as opposed to in tests). One of the big advantages to testing in the online world is that you can usually revert back to your original pretty easily.

How Do Companies Use A/B Testing?

Fung says that the popularity of the methodology has risen as companies have realized that the online environment is well suited to help managers, especially marketers, answer questions like, “What is most likely to make people click? Or buy our product? Or register with our site?” A/B testing is now used to evaluate everything from website design to online offers to headlines to product descriptions. (In fact, last week I looked at the results of A/B testing on the language we use to market a new product here at HBR.)

Most of these experiments run without the subjects even knowing. “As a user, we’re part of these tests all the time and don’t know it,” Fung says.

And it’s not just websites. You can test marketing emails or ads as well. For example, you might send two versions of an email to your customer list (randomizing the list first, of course) and figure out which one generates more sales. Then you can just send out the winning version next time. Or you might test two versions of ad copy and see which one converts visitors more often. Then you know to spend more getting the most successful one out there.

What Mistakes Do People Make When Doing A/B Tests?

I asked Fung about the mistakes he sees companies make when performing A/B tests, and he pointed to three common ones.

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First, he says, too many managers don’t let the tests run their course. Because most of the software for running these tests lets you watch results in real time, managers want to make decisions too quickly. This mistake, he says, “evolves out of impatience,” and many software vendors have played into this overeagerness by offering a type of A/B testing called “real-time optimization,” in which you can use algorithms to make adjustments as results come in. The problem is that, because of randomization, it’s possible that if you let the test run to its natural end, you might get a different result.

The second mistake is looking at too many metrics. “I cringe every time I see software that tries to please everyone by giving you a panel of hundreds of metrics,” he says. The problem is that if you’re looking at such a large number of metrics at the same time, you’re at risk of making what statisticians call “spurious correlations.” In proper test design, “you should decide on the metrics you’re going to look at before you execute an experiment and select a few. The more you’re measuring, the more likely that you’re going to see random fluctuations.” With so many metrics, instead of asking yourself, “What’s happening with this variable?” you’re asking, “What interesting (and potentially insignificant) changes am I seeing?”

Lastly, Fung says that few companies do enough retesting. “We tend to test it once and then we believe it. But even with a statistically significant result, there’s a quite large probability of false positive error. Unless you retest once in a while, you don’t rule out the possibility of being wrong.” False positives can occur for several reasons. For example, even though there may be little chance that any given A/B result is driven by random chance, if you do lots of A/B tests, the chances that at least one of your results is wrong grows rapidly.

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This can be particularly difficult to do because it is likely that managers would end up with contradictory results, and no one wants to discover that they’ve undermined previous findings, especially in the online world, where managers want to make changes — and capture value — quickly. But this focus on value can be misguided, Fung says: “People are not very vigilant about the practical value of the findings. They want to believe that every little amount of improvement is valuable even when the test results are not fully reliable. In fact, the smaller the improvement, the less reliable the results.”

How Not To Run A B&b

It’s clear that A/B testing is not a panacea. There are more complex kinds of experiments that are more efficient and will give you more reliable data, Fung says. But A/B testing is a great way to gain a quick understanding of a question you have. And “the good news about the A/B testing world is that everything happens so quickly, so if you run it and it doesn’t work, you can try something else. You can always flip back to the old tactic.”