Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, October 7, 2017

Preparation of Phosphate Buffered Saline (PBS)


While not commonly used in liquid chromatography, PBS solution is commonly used in preparing samples. By popular request, I am provided a common laboratory recipe for the solution here.

To make Phosphate Buffered Saline (PBS) solution:

Method #1:

1. To a 1-liter flask, add the following four anhydrous salts:

a. 200 mg KCI

b. 8,000 mg NaCI

c. 200 mg KH2PO4

d. 150 mg Na2HPO4

2. Add about 850 ml of deionized water and stir to dissolve the salts. When fully dissolved, fill to the “line” with more deionized water. Stir a final time to insure a uniformly mixed solution.

3. Pour the contents into a laboratory beaker and adjust the pH to 7.0 with 10% phosphoric acid (phosphate solutions should be adjusted with phosphoric acid only).
4. Filter the final solution through a suitable 0.22 micron filter before use.

Method #2:

Optionally, use the same ingredients as specified above, but premix in a beaker with stir bar to make the job easier. 

Place a 1 L laboratory glass beaker on a hotplate stir with stir bar. Fill the beaker with 850 ml of deionized water. Stir at a moderate rate with some heating (~ 35C). Add the dry ingredients to the solution and allow time for them to dissolve. When fully dissolved, remove the stir bar, remove from the heat and carefully pour the contents into a 1 L volumetric flask. Allow the solution to cool to ~ 20C. Fill to the line with deionized water, stopper and mix the final solution (inversion). Pour the contents back into a laboratory beaker and adjust the pH to 7.0 with 10% phosphoric acid (phosphate solutions should be adjusted with phosphoric acid only). 

Filter the final solution through a suitable 0.22 micron filter before use.

Saturday, September 2, 2017

HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties

Widely used in RP HPLC method development, Acetonitrile (ACN) and Methanol (MeOH) are the two most common solvents you will use to develop methods. So, besides the fact that Acetonitrile is well know to have a higher elution capacity than Methanol, what other properties should chromatographer's be aware of? Let's discuss a few that all chromatographers should know.

First, a few comments about the preparation of mobile phase solutions. 

     There are two common methods of preparing binary mixture, V/V, mobile phase solutions.
  • Method #1 is to fill a volumetric flask with a specific volume of the "A" solution, then fill the flask up to the line with the "B" solution.
  • Method #2 is to fill a graduated cylinder (or volumetric flask) with a specified amount of "A" solution; fill a second graduated cylinder (or volumetric flask) with a specified amount of the "B" solution and then mix the contents of both together.
Whichever method you use, please fully document it in your HPLC method so anyone reading it will be able to accurately reproduce it. The two methods described above are both correct in design, but will result in solutions with different properties.

For HPLC grade solvent (*we should always use HPLC grade solutions in HPLC analysis) ACN has the lowest absorbance (~ 190 nm) of the two making it well suited for low UV analysis. MeOH has a higher UV cut-off around 205-210 nm, slightly limiting its use in the very low UV ranges.

There is a significant difference between ACN and MeOH in their ability to dissolve many types of buffer salts AND samples. These differences are critical in method development. 

Solubility of the Mobile Phase:
  • A common reason for gradient runs to show poor reproducibility or to fail can be associated with running high concentrations of buffer with high concentrations of organic solution. While aqueous / organic solutions containing salt solutions of less than 10 mM concentration are not likely to precipitate under most gradient conditions (running to a max of 98% organic, not 100%), most buffer solution used with HPLC applications will have higher salt concentrations which may precipitate out of solution (resulting in clogs, leaks, plugs and inaccurate results) when the analysis conditions contains high percentages of organic solvent. Be cautious when selecting organic composition values in RP methods. Make sure the solutions used will be stable under all concentrations used. Also verify that the buffering capacity is still present when high organic concentrations are used (as your buffer is diluted). *Not sure if the salt will stay in solution? Just mix up a sample at the same concentration for a test. Look at it. Is there any turbidity or particulate visible? You have your answer.
  • Methanol's overall better solubility characteristics (better than ACN) mean that it does a better job of dissolving most salts (esp NH4, K and Na) at higher concentrations resulting in better performance and less precipitation.

Solubility of the Samples (effect on Peak Shape & Retention):
  • A fundamental requirement of liquid chromatography is that the sample fully dissolves in the mobile phase (initial mobile phase). Dissolve the sample in the mobile phase or in a slightly weaker strength solution (not a stronger solution) before analysis. This insures it will be loaded onto the head of the column as a concentrated slug improving peak shape and RSD. If the sample does not fully dissolve in the mobile phase then you are not in fact analyzing the whole sample. Another area where Methanol may be superior to ACN can be found in its ability to fully dissolve more types of samples. This improved solubility may result in better overall peak shape. Methanol also has different selectivity than ACN (not just the elution strength) which may result in peaks eluting at different retention times than expecting. This is another reason why we always try different mobile phase mixtures containing either ACN or MeOH when developing RP methods. Never assume that one solvent will be better than the other. Too many novice chromatographer's use only ACN as their main organic solvent for method development. Please don't make their mistake as such a strategy indicates a lack of practical experience and knowledge. You must first try them both separately (ACN & MeOH) to evaluate the results with your sample (best to start with comprehensive gradients at different pH values, as applicable). You will be rewarded for putting in the initial time to test both types of solutions as no simulator has yet been developed which can predict a truly accurate result with your own sample(s). You may be surprised to learn how many samples show better peak shape and performance using MeOH solutions. If no improvement is seen, well at least you now know this because you tried it and can move forward with confidence.

  • ACN is less viscous than MeOH so will usually result in lower column and system back-pressures overall. Mixtures of ACN and Water will also exhibit an endothermic reaction (cooling the solution) which can trap gas inside the solution. If you pre-mix your mobile phase, let it rest for several minutes after preparation.
  • MeOH is more viscous than ACN alone. It also has an unusual property where a 50/50 mixture of MeOH and Water will result in a much higher system and column back pressure than either MeOH or Water alone will. The effect is very Gaussian with a peak pressure observed with a 50/50 mixture. An exothermic reaction also results from an initial mixture of the two solutions giving off some gas. When preparing solutions it is best to allow the solution to rest for a few minutes to out-gass before topping off or using in the HPLC system.

I hope that this short discussion about some of the differences between these two popular HPLC solvents will aid you in developing better quality HPLC and LC-MS methods.

Reference: Table of HPLC Solvent Properties

Saturday, July 8, 2017


When selecting standards for use in testing an HPLC column OR for evaluation of an HPLC system’s performance (e.g. System Suitability, Performance Verification, Retention), in addition to selecting special high purity chemical compounds, consider using the actual sample which is specific to the method or application. Characterize the sample’s retention, peak shape and spectra (if applicable) and use the data to measure, compare and detect changes in  performance over time. 

Running Sugars on an Amino Column? Use simple and complex sugars as standards. Example: D-Fructose; D-Glucose; Lactose; Maltose; Sucrose.

NP (prepare the test solution in a mixture of Hexane/Ethanol, as appropriate)
Diethyl phthalate; Dimethyl phthalate; Toluene; Benzene.

RP (prepare a test solution in ACN/Water or Methanol/Water, as applicable)
Select 4 or 5 of these compounds for use in a mix. Uracil; Benzene, Acetophenone; Toluene; Naphthalene; N,N-Diethyl-m-toluamide; Phenol;  diethyl phthalate; diamyl phthalate; di-n-hexyl phthalate; dioctyl phthalate.

Include a Void Marker in your Test Solution:
Always measure the actual void volume of your specific HPLC column with a compound which is un-retained by your column. For RP applications which utilize at least 20% organic, Uracil or Thiourea are often used, but some inorganic salts (e.g. sodium nitrite and sodium nitrate) have also been shown to work as well.

Saturday, May 20, 2017

Chromatography Basics - Coffee Filter Chromatography

This month I would like to promote sharing and learning about chromatography with others. As scientists, sharing and passing on information to others is key to introducing new people to how we use science and the scientific method to explore what things are made of. Examples of chromatography are often seen in every-day life. For example, a spilled liquid drink on a napkin often reveals different colored or toned rings radiating out, hinting at the many compounds present within the one solution.

One of the simplest examples of a paper chromatography method can be set up and safely run at home. A jar with some water serves as the mobile phase and a coffee filter as the "Column" or support. Ordinary ballpoint pen ink (i.e. blue) or a magic marker can provide "samples" to evaluate. If available, various pure alcohols can replace the water and used as mobile phase solution(s) which may improve resolution. 

Over the past decades I have used these simple setups to demonstrate both what the analytical technique of chromatography is and how it works to hundreds of people of all ages and backgrounds. Many of examples can be found on the web and I have included a link below to one of the more recent published methods from Scientific American magazine. Please demonstrate and share it with others. Perhaps in doing so you will unleash the curiosity of someone to delve deeper into understanding what things are made of or the scientific fields?

  Link to an article illustrating how to run some paper liquid chromatography experiments at home. "Chromatography: Be a Color Detective"

Saturday, April 8, 2017

LC-MS Contamination? Another Possible Cause. Are your Mobile Phase Bottles and Filters Clean ?

One of the more common LC/MS problems I am asked to help solve deals with contaminated LC-MS or LC/MS/MS systems. Over time, many systems will become contaminated with a wide variety of plasticizers, detergents, salts, metals and ion pairing agents that routine source cleaning will not remove. Often, these compounds are introduced to the system through the tools used (e.g. pipettes) chemicals, solvents, mobile phase additives or even the samples themselves. "Dirty" samples sometimes persist inside the system long after the analysis work is complete, leaving material in poorly maintained injection valves but also through the use of poorly washed / contaminated and fouled HPLC columns. Even the modern inline HPLC vacuum degasser has proven to be a source of contamination. 

In addition to the above mentioned sources of contamination, another more obvious source of contamination should always be addressed early in the process of cleaning the system. Specifically, the glass mobile phase bottles and the associated solvent pickup filters used with them. Contamination in these areas directly infuses the system with undesirable material, so good practices must be maintained to reduce this source of potential contamination. 

As a general guideline, we should not place our mobile phase reservoir bottles in any type of dishwasher or wash them using any soaps. These will leave a residue easily detected by even the weakest mass spectrometer. Avoid contamination by purchasing high quality glass bottles with vented caps to keep dust out. If rinsing with organic solvents (and/or freshly prepared and filtered high resistance water) does not clean them, you can try a Nitric Acid rinse (30%) followed by a neutralizing wash in 2M Sodium hydroxide. Follow-up with a few rinses of HPLC Grade water (or LC/MS grade) then re-fill with an appropriate mobile phase. Don't forget to replace those solvent pickup filters too. Most of the sintered glass style filters are designed to be disposed of (not cleaned or put in an ultrasonic cleaner!) so dispose of them and install new filters and fresh mobile phase into those recently cleaned bottles before you start looking for the source of contamination in the more expensive parts of the instrument. - Please don't re-contaminate an expensive HPLC or LC/MS system and your data because you skipped replacing a $10 part. Keep commonly used spare parts around and always maintain a clean system.

Saturday, March 4, 2017

The Three Most Common HPLC Questions and How To Solve Them

The three most common HPLC related questions I am asked each week can be summarized below. Test your basic chromatography knowledge. Before reading the answers, see if you can answer them correctly on your own.

  • "What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?"
  • "How Should I Wash or Regenerate My HPLC Column?"
  • "How Can I Tell if the Sample is Retained On the HPLC Column? or What Does It Mean When No Chromatography Took Place?"

Let us address each question in order and attempt to provide accurate answers (I have included links after each question to articles with more detailed explanations).

What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?
  • Retention times must be reproducible from run to run.The causes of an unstable baseline and/or changing peak retention time(s) are often related. Common reasons include: Column temperature fluctuations, inadequate mobile phase mixing or degassing, leaks, dirty column, sample overload, lack of pH or buffering control (weakly ionizable samples can be very sensitive to changes). *Full Article link with detailed answers, here.

How Should I Wash or Regenerate My HPLC Column?

Note: Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide or booklet which comes with the new column. Additional information can be found on the vendor's website or by contacting them directly.
  • Two issues must be addressed to answer these types of questions. (1) Always wash your column with a specific column wash solution which is stronger than your analysis solution. The use of a stronger solution (In this context, "stronger" means better at dissolving the samples and faster at eluting them from the column) as the wash solution requires regular use to maintain the column. Failure to regularly wash your column may result in compounds accumulating on the column over time (fouling the column) resulting in poor reproducibility, higher back-pressures, contamination and/or poor peak shape. (2) Next, always wash your column after each analysis. This should be a separate step, not incorporated into your analysis method. The analysis method should not include the column re-equilibration steps at all. A second, separate wash method should always follow each analysis method which includes the rinsing of the column with a "stronger" solution for an adequate period of time, then adjustment back to initial conditions where re-equilibration can take place to get it ready for the next analysis run. These are fundamental guidelines of good method development and follow well established principles. Developing methods in this way should increase the lifetime of your columns and improve the reproducibility of results obtained (better %RSD run-to-run).
For more information on washing bound proteins off RP HPLC columns, please refer to this linked article found here.

How Can I Tell if the Sample Is Retained On the HPLC Column? or What Does It Mean When the Sample Comes Out At or Near the Column Void Volume?
  • Chromatography is a tool which when used properly adds one or more additional dimensions of physical or chemical characterization information to your analysis data. It does so first by using on-column RETENTION. Samples must be run under conditions which allow the material to interact with the chromatography support for a period of time. We define this time as the retention time. A sample which does not interact at all with the column support material will elute off the column early (and not be retained) at the "column void time" (or column dead time). We refer to this void time as the "T zero" time. When a sample elutes at or near the T zero time, no chromatography has taken place and no method has been developed. It is as if the HPLC column was not used. How do you know what the "T zero" time is (it will be different for different methods)? You must first calculate the HPLC column's dead volume. Once you know the column dead volume and flow rate, you can calculate the T zero time. A scientifically valid HPLC method will include conditions which retain the sample on the column for a long enough period of time to insure that it is interacting with the support. This allows for separation from other compounds to take place and is the purpose of chromatographic resolution. Without this retention mechanism, you are just flow-injecting the sample past the column and skipping all chromatography. It would be far simpler to just place the sample in a spectrophotomer cell as no retention or additional data would be obtained using that technique.
  • When first learning liquid chromatography, two of the very first calculations you must learn to use in HPLC are: Column Dead Volume (aka: Column Void Volume) and the K prime of a sample (aka: Peak Capacity Factor). Do you know how to calculate these? They are calculated and reported for each method used. You should be able to tell anyone who asks you what the values are for each method. A chromatographer must know and understand them before using an HPLC system or running a method. They are also critical to method specificity and proper validation. Here are links which after reading and practicing, should make you an expert in these two fundamental calculations. 

So, how did you do answering these basic questions? If you have put in the needed study time and practical experience to learn and use these fundamentals of high-performance liquid chromatography, then you should have been able to easily provide correct answers to all three questions. If not, then it is time to go back and study up on those basic liquid chromatography texts and article links, plus get more supervised hands-on time with the instruments.