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. 

"Determination of HPLC Column Dead Volume / Dead Time (T zero)";

"K Prime (also known as: Capacity Factor or Retention Factor): One of the Single Most Important HPLC Parameters of All".

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.

Typical Commercial Strengths of Common Acids and Bases Used in HPLC

CHEMICAL NAME	MOLECULAR WEIGHT	MOLES / LITER	GRAMS / LITER	PERCENT by WEIGHT	SPECIFIC GRAVITY
Acetic Acid	60.05	6.27	376	36	1.045
Acetic Acid, Glacial	60.05	17.4	1045	99.5	1.05
Formic Acid	46.02	23.4	1080	90	1.21
Hydrochloric Acid	36.5	11.6	424	36	1.18
Nitric Acid	63.02	15.99	1008	71	1.42
Perchloric Acid	100.5	11.65	1172	70	1.67
Phosphoric Acid	98	14.7	1445	85	1.70
Sulfuric Acid	98.1	18.0	1766	96	1.84
Ammonia (in H20)	17.0	14.8	252	28	0.898
Potassium Hydroxide	56.1	13.5	757	50	1.52
Sodium Hydroxide	40.0	19.1	763	50	1.53

Data obtained from The Merck Index, 11^th edition (1989).

HPLC Column Support Pore VOLUME

If an HPLC column had no packing material inside it, then the volume of liquid contained in the cylinder could be calculated using the formula for the volume of a cylinder as follows: 

      Volume of Cylinder = Pi * r² * L;     

          [where Volume is in ul; Pi = 3.14; r = column radius (mm) and L= column length (mm)]

  Example: Using the above formula, a 4.6 mm x 250 mm column would have an empty volume of 4,155 ul (~ 4.16 mls).

For most chromatography applications we pack the column with a high surface area porous media. Often this is a silica based support. This support media fills the empty space inside the column reducing the total volume accessible by a liquid (or to the samples). If the media used was not porous, it would fill most of the space (depends on size and shape of media). Most commonly used chromatography supports are porous and leave about 70% (0.7) of the original volume available to the mobile phase and sample [Pore Volume = Surface Area (m²/g) x Pore Diameter  (Å) / 40,000]. Based on this information, we use a value of 0.7 as the average pore volume for a packed chromatography column (some supports will have pore volumes which are larger or smaller than this value. The manufacturer will often measure it and provide the value on their published specification sheet).


Using a typical 4.6mm x 250mm column we found the total volume to be 4,155 ul (4.16 mLs). If we now multiply this empty column volume by 0.7 (note: use 0.7 or 70% for columns with fully porous particles and 0.55 or 55% for superficially porous particles) we obtain 2,908ul total volume (2.9 mLs). This is the estimated volume of the fully packed column. This value is very important as it provides an estimate of what the column dead volume will be so we can calculate the 'T' zero time of an unretained analyte. This estimate will depend on the column dimensions, using our HPLC method (be sure and take into account the measured flow rate to determine the column "dead time"). This is one of the very first calculations you make when starting or modifying an HPLC method and is critical information to know at all stages of method development. All chromatographers should know how to estimate this value before using an HPLC system. *You should confirm this estimate by injecting an unretained sample onto the column and measure the retention volume, then compare the two values. The measured value is the most important number (the one we use for calculations), but the estimate should be close (+/- 15%). The estimate is still useful for troubelshooting and method development as when combined with K prime, it provides a quick measure if chromatography has occurred (retention).

For more information on the importance of knowing the HPLC Column Dead Time, please refer to this article link. 

Notes: The measured support pore Diameter (SIZE) is important for determining if the sample will have access to the inside of the support (e.g. A support with a pore size of 80Å will be too small for most large peptides or proteins, but a support that is 300Å will allow access to many, not all, larger molecules). A support with too small a pore diameter will not allow the sample to access the high surface area inside the support. Instead, the sample will be unretained and pass by it eluting at the column's void volume. This is the basis of SEC or GPC analysis where we use columns with different pore sizes to "filter" samples based on size. Large pores for large Mw samples and small pores for low Mw samples. A general rule is use 300Å or larger pores for samples with Mw > 10,000 and 80Å to 150Å for smaller samples.

More info on pore volume can be found at this article link: https://hplctips.blogspot.com/2014/12/hplc-column-pore-volume-or-pore.html