Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)

   Let's take a quick look at two different problems which you may encounter when operating an HPLC system. We start with the basic observation and then look at the most likely causes so we can begin the troubleshooting process and repair the problem. An automated HPLC system's flow path typically consists of: The Solvent Pickup Filters (in the mobile phase reservoirs); The Pump(s); AutoSampler; AutoInjector; Column and one or more Detectors.*You should have a good understanding of this flow path before you proceed to diagnose the problem(s).

 *A gradual increase of pressure for the same method over time is often due to column fouling or a dirty inlet frit (e.g. PTFE frit). This article specifically focuses on the causes of a sudden change, not a slow change over time.

   Sudden System Back Pressure Changes: We will assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the back pressure has changed from what is normally seen. The problem must lie within the flow path of the system.

   Excessive High Pressure: Typical reasons for this are:

1.      A fouled or plugged column;
2.      Wrong flow rate (higher than normal);
3.      Inlet frit/filter plugged or restricted;
4.      Plugged line;
5.      Wrong mobile phase composition.

   Large Drop in Pressure: Typical reasons for this are:

1.      A leak at a fitting, column or line (Number one reason);
2.      Wrong flow rate (lower than normal);
3.      Wrong mobile phase composition. 

• Start by checking the method parameters to insure that they have not changed (i.e. flow rate, mobile phase composition). Check for leaks or plugs. If the column is suspect, replace it with a zero dead volume union (ZDU) and restrictor and flush the system. Replace the column with a new one or wash the current column according to the column manufacturer's guidelines.

   Sudden Peak Shape Changes: We will again assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the peak shape of one or all of the peaks has changed from what is normally seen. *The key thing to keep in mind is that the change occurs all of a sudden, not because of poor initial method development.

   Typical reasons for this are:

1.      Tailing or Split Peaks: Sample overload, change in flow rate, mobile phase composition (e.g. composition or pH), void formation, dirty frit, injection solvent too strong or a fouled column.
2.      Fronting: Commonly seen when overloading sample on column.
3.      Ghost Peaks: Usually due to a contaminated mobile phase, contaminated sample vial or contaminated injector (e.g. rotor seal).
4.      Broad Peaks: Large sample injection volumes or extra column volume (bad connections with the system or tubing) are usually to blame. Try reducing the injection volume by a factor of 10 and see if the problem goes away. You may also want to wash the column as it may be fouled with sample.

   These are just two common problems we see when using HPLC systems. Note that a dirty or fouled column can cause many of these problems so take care of your columns and wash and test them regularly to insure they are in compliance. There are many other commonly seen problems besides these. If you would like to see a specific problem featured on this blog, then please send me a request.



  

HPLC Maintenance & Repair Parts To Have on Hand for HPLC Systems

HPLC (UHPLC) systems are complex instruments which require periodic inspection, cleaning and maintenance. These tasks are critical to maintain the performance, reliability and accuracy of the instrument. If you have not done so already, I strongly recommend that you create formal standard operating procedures (SOP's) which address: (1) The frequency of when routine and non-routine maintenance procedures should be performed; (2) The types of maintenance and/or repair procedures used (e.g. piston seal replacement, A/I rotary valve seal replacement); (3) The exact step-by-step procedure to follow in performing these tasks and (4) The Performance Verification or Qualification steps and procedures which are to be performed to verify that any repairs made have been done correctly. *An instrument log book should be employed to document these procedures over time.

Periodic "General Maintenance" of the HPLC is one type of service procedure which should be scheduled at a set frequency (Example: Every 6 months) and will serve to provide a time to clean, inspect and repair/replace any parts which are worn due to normal use. Such routine HPLC maintenance is often referred to as a basic "Preventative Maintenance" service (or "PM Service"). Spare parts common to your HPLC system(s) should be on hand to perform these scheduled maintenance procedures as part of a normal PM service.

Here is a list of common parts that should be on hand for a "typical" HPLC system used in a pharmaceutical laboratory. Please consult the appropriate manufacture's product literature to determine the correct parts needed for your own HPLC system. This list is presented as a general guideline only:

• Capillary tubing, fittings (nuts and ferrules): Assorted fittings, usually made of 316 Stainless Steel, but could be made of polymeric materials. Always have spare precut and polished chromatography tubing of appropriate I.D. and lengths for use with your HPLC available at all times. Insure that the nuts and ferrules used are appropriate for your brand of HPLC system and the columns used as different manufacturers have different specifications for their fittings and ferrules. Many types are not interchangeable.

• Detector Lamps: At least one spare bulb of a type designed for your specific detector should be on hand. Note that some detectors use multiple lamps so you may need to have more than one type available for each detector. Some lamp bulb types (e.g. tungsten) can be safely stored and last for several years while other types, such as Deuterium bulbs, loose substantial energy after as little as 6 months. If you have several detectors of the exact same design, then there is often no need to stock multiple replacement bulbs for each one. Instead, stock enough bulbs to service one detector as it is unlikely you would see failure of more than one detector on the same day (an exception to this guideline is if you perform PM services on all of the instruments at the same time, then you may want to have multiple bulbs available).

• Pump Pistons: One set of spare new pistons should be kept on hand for each pump module. As with lamp bulbs, if you have several identical pumps, then there is often no need to stock multiple sets of pistons for each one. Stock only as many as you expect to use in one year. Clean and inspect the pistons during each PM for any signs of scratches or surface abrasions. Under routine use, pistons should only require general cleaning and last a long time before replacement is required (> 1 year). Mobile phases which contain high concentrations of salt buffers often accelerate this wear requiring more frequent replacement. *Always install new piston seals when replacing pistons.

• Pump Piston Seals: At least one set of spare new piston seals should be on hand for each pump module. Seals wear out more frequently than pistons. You should go through two or more sets of piston seals before you need to replace the pistons. If the piston seals leak, inspect the pistons for wear (replace with new ones or clean and reuse) and install new piston seals. Mobile phases which contain high concentrations of salt buffers often accelerate this wear.

• Solvent Pickup Filters: These are the large particle filters which sit inside your solvent or mobile phase bottles. They are often made from stainless steel or sintered glass with porous inlets (~10 to 30 micron) and can clog or become fouled over time (esp. when used with aqueous buffers). In some cases these can be cleaned using sonication (not sintered glass filters, only steel or polymeric!). Note: Sometimes it is most cost effective to replace them with new filters then clean and re-use them.

• Inline Frits/Filters: You may have an inline filter placed after your PUMP head, but before the column inlet to collect any remaining particulate matter. These filters can extend the lifetime of the entire HPLC system (esp. the A/S, A/I and Column), but will only do so if changed on a regular basis. Some manufacturers incorporate this type of filter into the design of their pump modules. An example of this can be found on the HP/Agilent brand model 1050, 1100 and/or 1200-series pumps. These have an inexpensive 10 micron PTFE frit installed in the outlet valve of the pump. This filter catches all of the normally occurring piston seal debris and larger mobile phase particles and should be changed every month. Other pre-filters are installed in cartridges just before the column inlet. These often overlooked pre-filters filters must be replaced about once each month to do their job properly. Keep plenty of spare filters on hand.

• Auto-injector Rotary Valve Seals: If you have an auto-injector, then a high pressure valve is probably used to switch the sample into the flow path for analysis. This valve will have one or more parts which require regular inspection, cleaning and periodic replacement. Mobile phases which contain high concentrations of salt buffers often accelerate this wear. The valve rotor seal is the most common part which requires replacement.

• Auto-Sampler Needle: A needle should last a very long time, but depending on the frequency of use and type of vial septa encountered it can require replacement at regular intervals. A good general guideline would be to keep one spare needle on hand for every 2-4 systems.

• Auto-Sampler Needle Seat: The needle seat often requires more frequent replacement than the needle due to repeated mechanical wear. A good general guideline would be to keep one spare needle seat on hand for each system.

• UV/VIS Detector Flow Cell: While not actually a required PM spare part, this one is worthwhile to have. If you employ a UV/VIS flow cell, then I always suggest you keep one dedicated spare flow cell on hand which matches the size and volume of the type you use in your instrument. A spare flow cell can prove to be very valuable as a troubleshooting tool if you believe that you have contaminated or clogged your current flow cell. A quick swap can answer the question and get you back to work quickly saving hours or days of lost time. *Note: This extra flow cell should be kept separate from all instruments for use as a tested spare only and not used for regular analysis.

If you have suggestions for other types of common HPLC spares to add to the list or to have on hand, then please let me know.

What type of Water Should I use for HPLC, UHPLC or LC/MS Analysis?

Water is one of the most common solvents used in reversed phase chromatography. HPLC and LC/MS work demands ultra pure quality water be used in all applications which call for it as part of the method. Special types of HPLC analysis, such as amino acid analysis and ion chromatography, demand fresh ultra high quality water be used or artifact peaks may result. Poor quality or low grades of water may lead to "ghost peaks", baseline instability, high background noise or signals, contamination of columns and an inability to obtain reproducible results. Use the freshest and highest purity of water for best results.

A good starting point for describing the type of water suited to liquid chromatography applications is to look at the specification for ASTM Type 1 Reagent grade water. We often exceed this requirement for chromatography applications as several unspecified items such as nitrates and other chemicals present may have a negative effect on our analysis methods.

How does the grade of water affect our chromatography? The grade specified often dictates the amount of organics, bacteria, particulate, residues and overall absorbance the water will have. For example.

(1) Organics: High levels of T.O.C. can accumulate on the particles, inside the pores, or bind to active sites on the support inside the column causing a loss of resolution or sensitivity. *Lower T.O.C. levels are desirable.

(2) Bacteria: Microorganisms can contaminate the buffer solutions used causing ghost peaks, column fouling and the release of additional foreign organic matter into the system. This can result in clogs, ghost peaks, poor reproducibility or loss of resolution and/or sensitivity. *The water should be filtered through a 0.2 micron filter before use. Refrigerate solutions for no more than 3 days to slow growth, then dispose of the solutions.

(3) UV absorbance: High background or interfering ions which absorb can raise the baseline and noise levels seen, decreasing the total dynamic range. *Again, the lowest values, esp. at 200nm, are desirable.

A few of the general requirements for HPLC grade ultrapure Type 1 water can be stated as follows:

   Resistivity :         > 18 MΩ•cm at 25.0 C
   T.O.C. :              < 5 ppb
   UV cutoff :          190nm (as low in absorbance as possible!)
   Filtered :             0.2 micron Filter

*Some suppliers will also specify residue after evaporation (usually < 2 ppm); Trace metal analysis; Optical properties at specified wavelengths and other information. If purchasing by the bottle, request a copy of the lot certification sheet for the water so you can compare the measured values to other products.

Generating your own in-house, reverse osmosis (RO) ultra pure water from potable tap water is one of the best ways to insure you have high quality water for your LC methods. These systems pre-filter the water to remove large particulates then typically use UV lamps and/or multiple resin cartridges to remove the maximum amount of T.O.C.'s from the water plus many trace metals before finally filtering the water through a 0.2 micron membrane as a final polishing step. Various types of systems can be purchased, but for HPLC or LC-MS applications, it is critical that you select a system that provides ultra pure water suitable for your applications. Periodic maintenance of the filter cartridges and monitoring of the main water supply source is critical to their operation (some "tap" water sources may require pre-treatement). *"Water On Demand" systems such as these provide fresh clean water on demand so there is no need to be concerned with storage issues. A number of different vendors offer these lab grade systems for HPLC and LC/MS applications and you can contact them (e.g. Millipore/Sigma Milli-Q® brand) to determine which system will provide you with the volume and quality of water which is appropriate for your application(s).

If you do not have access to an in-house reverse osmosis system, then purchasing HPLC or LC/MS grade water in glass bottles may be another option. A hint, before opening and using them,  clean the outside of bottles of all dust. Date the bottles when you first open them. Bacteria will start to grow once the bottle has been opened. The glass will also slowly leach ions (i.e. Sodium) over time into the water so it is best to use the water quickly.

Never underestimate how the quality of the water you use to perform chromatography can change the results seen in your methods. Water quality is just as critical as any other component in your system so be sure and take the time to monitor it just like you do to any other part of the system.

Method Development Hint: Use your HPLC Diode Array Detector (DAD or PDA) as a Spectrophotometer

One of the many useful features of a UV/VIS scanning diode array detector is that it can be employed in flow injection mode to scan a sample and provide you with some useful data about the absorbance characteristics of the sample (which probably contains a mixture of components). Unlike a spectrophotometer, you only need about 1 ul of sample instead of a 1ml cuvette and only 15-20 seconds of time to gather the data.

Why do this? I use this feature often when I receive a new and unfamiliar sample for method development. I set up the detector to scan and store all wavelengths, in steps of 2nm, from 210nm to 450nm and inject the sample in flow injection mode (that means no-column is present and I easily do this using the By-Pass position on my column selector). In a very short amount of time I can view the resulting spectra of the sample which aids me in selecting the initial discreet wavelengths to monitor. For example: If I notice that the sample shows some absorbance at 410nm using the flow injection run, then notice while developing the analysis method that none of the peaks seen show absorbance near 410nm, then I can assume that I may still have some components retained on the column.

Setup Hints:
(1) For this to work well, you should have a high performance, low volume switching valve or automated column selection system (e.g. The LC Spiderling Column Selection System) installed so you can easily by-pass your column (otherwise, remove your column and place a high pressure, low volume union in its place).
(2) Set the diode array detector to a high sampling rate because the sample is going to fly through the flow cell quickly. Use a sampling rate that is faster than you would use if a column was there to disperse the sample and slow down the peaks.
(3) Choose a wide range of wavelengths to scan and store. If the sample appears colorless to the eye in solution and I am running in a UV transparent solvent such as acetonitrile, then I often use a range of 210 to 450nm.

Proper Wavelength Selection for HPLC Method Development (or Purity Determination)

Selecting the best HPLC wavelength(s) to monitor during an analysis method for use in quantitation and/or purity determination requires both knowledge and careful attention. Here is the basic procedure to use:

Step 1. Create the Method. To determine which UV/VIS detector wavelength(s) should be chosen for the analysis of your sample, you will first need to create a general HPLC Method which retains and resolves the compound(s) of interest on the column (goal is a K prime of >2.0, less than 10.0). Be sure and utilize a scanning diode array detector in full scan mode (often referred to as a photo-diode array detector, PDA or DAD) to scan all relevant wavelengths of your samples (e.g. 210 to 450nm). Note: Your choice of mobile phase and detector settings will effect the S/N values.

Step 2. Determine the lambda max of the sample's spectra using the Data analysis software. Once you have completed the analysis, review the spectral data to determine which prominent peak wavelengths have the maximum signal to noise (S/N) ratio. These “peaks” can be used as the individual wavelengths for integration and purity determination. By sure and double check that any detector options which use a “reference wavelength" are turned ‘OFF’ when running these methods (more info on “reference wavelengths” can be found on this blog in another post). With the wavelength selected, chose an appropriate bandwidth for use (narrow).

Step 3. Edit the HPLC method to use the discreet wavelengths found in step 2. Whenever you run a real sample, continue to use the full scanning mode of the detector so you will know about any other components which absorb at wavelengths far away from and/or near the peak wavelengths. These compounds can add or subtract signal from the main peak making it appear to be more or less concentrated (or more or less pure) than it actually is. If you only monitored the sample with a single wavelength detector, then you would miss this vital information and make errors in your purity or concentration determinations.

Conclusion. (1) Using a multi-wavelength, scanning HPLC detector such as a DAD is one of the most important tools you can use to create accurate and reliable chromatography methods. Always use a scanning DAD for method development to prevent errors. (2) Learning how to correctly use and set up the detector's settings, parameters, special features and options may prevent false or misleading results. Only after you have developed a reliable and repeatable method with good sample retention and peak shape can you begin to report accurate integration and concentration values (and/or make UV/VIS "purity" determinations).

Troubleshooting HPLC Injectors (Manual and Automated)

Sample injectors are a critical component of a chromatography system. Understanding how they operate as well as the proper techniques to use and maintain them are fundamental skills needed to operated an HPLC system. Lets briefly discuss some of these fundamentals as applied to a standard manually operated HPLC injection valve and also in an automated mode as found in an autosampler. *Note: You should always refer to the specific manufacturer's product specifications, operation, servicing documentation or support personnel before servicing any injector.

MANUAL INJECTION VALVE Notes:

These valves allow you to use a high precision syringe to manually fill a fitted "loop" with a sample and then, by turning a valve handle, introduce the sample to the high pressure flow stream directed toward the column inlet. Sample loops are available in a wide range of volumes and take just minutes to install. The injector valve has very tiny openings inside which are moved between two different positions (LOAD and INJECT). The LOAD positions allow the valve to seal off the internal high pressure flow from the loop to allow it to be safely filled with sample at atmospheric pressure. The INJECT position introduces the liquid contained inside the loop to the main flow path (under high pressure). The parts must be clean and seal well to insure proper function. Leaks from all areas (except the vent) are not acceptable and indicate a problem. Here are a few tips regarding the use of manual injectors in HPLC.

• Use the correct type of sample syringe. Usually these are high precision glass syringes with Teflon plungers. The needle tip style is the most critical item! Most injectors are designed to only work with a needle which has a squared off tip (NOT a point as is commonly used in GC!). The most common gauge used is #22. Always check with the valve manufacturer to determine the correct style and gauge of needle before use.
• Leave the valve in the INJECT position during the entire run to flush it clean of sample and stay equilibrated with your method. Switch it back to LOAD only when you are ready to load a new sample.
• For high reproducibility and accuracy within one HPLC system, fill the loop with at least three times the volume of the loop with sample to insure that the entire path is full of sample. This is known as the complete or over-filled loop method. *Choose your loop volume size with this in mind. Loading the same volume as the loop will often result in poor accuracy.
• Loops often do not contain the exact volume stated on them. They can be off by ~25% so consider this when injecting partial volumes and not using the standard over-filled loop method.
• Types of common leaks: (1) Leaks at the needle port (needle seal worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

AUTOMATED INJECTION VALVE (auto-injectors) Notes:

These valves use a high precision syringe or high pressure pump to fill a fitted "loop" with a sample and introduce the sample to the high pressure flow stream, all automatically. Most function exactly the same as described above, though some are based on true high performance liquid chromatography pumps so have no "syringe" at all (e.g. Agilent 1100/1200 designs) Here are a few tips regarding the use of automated injectors in HPLC.

• Regular maintenance is even more critical with auto-injectors since you often can not see what is going on during the injection cycle. Leaks, if present can be harder to find so make it a habit to visually check all of the areas around the injector regularly.
• Needle and Needle Seats are normal wear items on these instruments. As such, they require routine checking for leaks or damage and replacement when worn.
• Vial Caps: If you make multiple injections from one vial (or large volume injections) with a tightly sealed vial cap, a vacuum can form inside the vial causing volumetric errors to occur in your samples (resulting in you injecting less sample each time). Leave the caps slightly loose to avoid this problem. Multiple injections into the same vial can also cause the needle hole to become enlarge over time allowing the sample or solvent to evaporate over time, changing the concentration of the sample (more concentrated). Replace the cap and seal with a new one if used multiple times. Always leave the cap slightly loose.
• Loop volume: Autoinjectors often incorporate one large loop to handle a wide range of sample volumes. This is a trade off of accuracy for convienence. Accuracy is often poorest at the very low end of the range and best near the middle to high end. Always verify the reproducibility of the injector to inject a specific volume through statistical analysis of repetitive injections.
• Types of common leaks: (1) Leaks at the needle seat (needle seat worn); (2) Leaks behind the valve stator (worn rotor seal, buffer crystals dried inside, over pressured, scratch on rotor); (3) Leaks at the vent (liquid should be expelled from the vent when filling the loop only. Other leaks indicate a problem). Note: Rotor seal damage can cause sample carry-over problems so valves should be inspected at regular intervals (~ 6 months).

These are just a few tips related to HPLC injectors. Please consult the service documentation for your specific instrument to better understand how the system works and what areas you should be monitoring. Understanding HOW these systems operate (and can fail) is one of the most important skills you can learn as a chromatographer. Take the time to understand the complete flow path of your system before using it.

HPLC Solution Degassing, Sparging With the Wrong Gas (Gas Choice Matters)

The other day I took a call from a client whom explained they were having a number of problems with their HPLC pump. They felt that they were very experienced chromatographers whom had been unable to find the reason for why their pump flow stability was poor. It had very high ripple and noise. The pump had been fully serviced one month earlier and passed all qualification tests. Their UV/VIS detector appeared to work fine and was ruled out as being the problem early on. They used HPLC grade filtered solvents, operated at an appropriate flow rate, had a clean and tested column installed, always primed their pump before use each day and sparged each solvent reservoir with a low stream of continuous gas kept away from the solvent inlet lines. Everything seemed in order, but something was clearly wrong. Their vendor suspected the check valves were to blame so they purchased and installed new ones with no change. They still had an unstable flow rate under all conditions tested (pump pulsation of 5%). When I asked them if they had changed anything related to the HPLC system in the past few months I was reassured that nothing had been altered.


Often the best way to solve a problem is to start at the beginning. Take nothing for granted. This started as one of those many phone calls I receive where someone wants me to solve their problem over the phone and by not visiting their laboratory. Sometimes this is possible, but sometimes the problem is something that can only be seen by being physically present in their laboratory. I felt this was one of those times. So, they agreed to pay for a few hours of consulting time to have me come out and go over their system to find the problem. Once I arrived at the client's lab I quickly went over to inspect the layout of the equipment and check the tubing connections for the correct fittings and tightness. Next, I looked at the software parameters being used to operate the system. Some small issues were found, but not enough to explain the problem seen. I then looked at the physical output of the pump and detector to get a better idea of the period, cycle and type of noise seen. While I was reviewing the data and still looking over the system, I found the problem. The high pressure gas cylinder next to the instrument was labeled ARGON. Argon was being used as the sparging gas for the mobile phase instead of the more appropriate gas, Helium. They had in fact recently switched to argon gas because it was less expensive to use than helium. The person (their senior chemist) who had made this substitution was rewarded for his cost-cutting suggestion. Their choice of argon gas had of course cost them several weeks of down time while they tried to solve this problem on their own.... not much of a savings when you consider that! So, they had in fact caused the problem themselves, but were not aware of the fundamental reason why changing to argon gas was a very bad idea.

Why does the gas choice matter? For liquid chromatography applications we only use high-purity helium gas for sparging because it is one of the few inert gases which is the least soluble in water and mobile phase solutions. Gases such as argon and nitrogen ARE soluble in water and mobile phase solutions.  While they can be used to displace oxygen from air (great if you are making wine, but not so great if you are using the solution for HPLC), they infuse the liquid with gas (like a soda). Helium easily displaces air (oxygen and nitrogen) from solutions while not adding significant amount of dissolved gas to the solution. Helium is the least soluble and most inert gas to use. If we sparge with argon or nitrogen, then we infuse the solution with gas. This is the opposite of what we wish to accomplish by degassing our mobile phase. Please, if you wish to use the continuous gas sparging method to degass your mobile phase, then use high-purity helium gas only.

So I suggested that they replace their high pressure argon cylinder with a tank of high purity helium. Luckily they still had their original helium tank available so we hooked it back up. I sparged their mobile phase with the helium gas for about ten minutes then primed the pumps with the solution. The helium was left continuously flowing at a very low pressure (~ 2 psi) through a dedicated SS frit in the mobile phase. This keeps the level of helium in solution constant over time, resulting in stable baselines. After about five minutes the pump was running smooth and about as pulse free as you could hope for (0.1% pulsation). Lesson: Never assume anything and don't forget to make decisions which incorporate some basic scientific reasoning into them first.

Using Smaller Diameter HPLC Columns (Calculate Linear Velocity)

Lots of 2.1mm ID chromatography columns are appearing on the market right now. Since most of us are using 4.6 mm ID columns to develop HPLC and UHPLC methods, use of these smaller ID columns requires a few adjustments be made to the method and often, the HPLC system. If gradient elution is used, then the gradient profile must be changed to compensate for changes in void volume of the column and the dwell volume of the system. Injection volume must also be adjusted in a linear fashion too. Additionally, to maintain the same initial mobile phase linear velocity through the column as we had before (to obtain the same approximate retention times), the flow rate must also be adjusted. *We will discuss how to calculate the change in flow rate in this installment.

In order to reproduce your original method, we must first adjust the flow rate for the new, narrower bore column. The formula to do this is very simple. We decrease the flow rate by using the square of the ratios of the column diameters times the flow rate.

Linear Velocity Change Formula:

( C₁  / C₂ )² x original flow rate (ml/min) = new flow rate (ml/min).

Where:  C₁  =  Diameter (mm) of new (smaller) column;
              C₂ =   Diameter (mm) of original column.
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Example #1: Find the new linear flow rate if we use a 2.1 mm ID column in place of a 4.6mm column with an initial flow rate of 1.000 ml/min.

              ( 2.1 / 4.6 ) ² x 1.000 = 0.208 (208 ul/min)


Example #2: Find the new linear flow rate if we use a 2.1 mm ID column in place of a 4.6mm column with an initial flow rate of 2.000 ml/min.

              ( 2.1 / 4.6 ) ² x 2.000 = 0.416 (416 ul/min)



Example #3: Find the new linear flow rate if we use a 1.0 mm ID column in place of a 4.6mm column with an initial flow rate of 1.500 ml/min.

              ( 1.0 / 4.6 ) ² x 1.500 = 0.071  (71 ul/min)



If we assume that the original flow rate is 1.000 ml/min then we can also use this table to get an idea of how the flow rate changes with decreasing column diameter (same particle size and support).

Column I.D. (mm)                 Flow Rate (ul/min)
            4.6                                          1,000
            2.1                                             208
            1.0                                               47
            0.3                                                 4
            0.15                                               1



Summary: Scaling down a method which was originally developed on a 4.6 mm ID column for use on a 2.1 mm ID column (with the same particle size) requires that the flow path of the HPLC system be optimized (reduced) to minimize diffusion and the flow rate reduced five time to achieve the same linear velocity. If the particle size is also going to be reduced from 5u to 2.5u or smaller, then increases in the flow rate may be considered to take advantage of the optimized plate counts using optimized linear velocities (which are much higher for smaller particles).

Introduction to Size Exclusion Chromatography (SEC HPLC)

Size Exclusion Chromatography. Often known as “SEC”.

Other names used to describe SEC:

• Gel Filtration Chromatography or “GFC” is a commonly used phrase when you are separating biological molecules in aqueous (or sometimes organic containing mobile phases). It is often described as a gentle form of chromatography leaving the protein or sample intact (*Proteins are one of the most common molecules separated using this technique, but if needed intact, must be kept away from denaturing agents). 
• Gel Permeation Chromatography or "GPC" usually refers to the separation of polymers using an organic solvent, but water soluble polymers are also applicable too.

Basic Principle: Used to separate molecules based on their molecular size in solution (as the primary mode of separation). The pore size and interstitial volume of a packed column must be determined to find out which molecules it excludes. Small molecules which are smaller than the pore size will enter the particles and spend more time navigating the channels within than larger molecules which will be excluded from entering the particles and exit quickly. It is extremely important to measure this so you know what the actual column volume is AND what the exclusion limit is. Manufacturer’s often report these exclusion limits via calibration tables for linear standards such as dextran or polystyrene though some provide data using globular standards which provides more accurate data when running many proteins or peptides. Please keep in mind that the actual confirmation (hydrodynamic volume) of the compound in the mobile phase may be different than what any of these standards are so the best column to choose may be one with a different pore size than suggested (this is why it is so important to test your compound on actual columns). Determine the actual exclusion volume running actual samples. They should elute at the Tzero point (column void volume).

Support Types: Available supports are most commonly based on either silica gel or polymeric materials (e.g. DVB). Their properties and chemical compatibility may vary so be sure to document which back-pressure ranges, pH, flow rates, temperature and/or solvents are safe to use with them.

Technique: Improved resolution often results from chaining columns together, in-series, with the same pore size. Additionally, a broader range (size) of molecules can often be separated using multiple columns with differing pore volumes together, in-series (very common in GPC applications). Single "Mixed Pore" columns are also available from many manufacturers which allow a wide range of molecular weights to be screened, though often at reduced resolution. It is important to make sure that there is no interaction between the stationary phase used and the solute employed to transport the sample. This will insure that the only mechanism being used is size exclusion.

Misc. Method Development Notes: 

(1) As the primary mode of chromatography is based on "size", achieving acceptable K prime values for retention are not applicable in this mode. K prime is NOT applicable to ion exchange or SEC modes. You must achieve retention past the initial pore exclusion point to demonstrate that the compound(s) are interacting with the pores of the phase. Measure the actual column volume to determine Tzero (this is very important). Inject an unretained compound to confirm and record the pore exclusion limit with a suitable high Mw standard.

(2) For silica based supports, strong salt buffers are often employed. You must insure proper miscibility of the sample and mobile phase at all times. Be sure and flush the system of all buffers at the end of each day. This is critical and not an optional step if you want to maintain the chromatography hardware. Salt crystals can be corrosive to the steel used in these system and may result in damage to the pump, injector and other components if not flushed out. Use a flushing solution that is similar to your mobile phase, but without the buffer. If you see any salt crystals forming on the instrument, then you have not been flushing the system down properly, or often enough. Salt should never be visible on the outside of the instrument. 

(3) Method development using buffered mobile phase solutions may employ several key variables to achieve good results. After selecting the correct column(s) use a linear flow rate and systematically adjust: (a) the molarity of the buffer salt used (e.g. 10 mM, 50 mM, 100 mM, 0.5M ...); (b) the pH of the solution (acidic, neutral, basic); (c) the temperature of the column to achieve satisfactory resolution. Note: Selecting the best column is the single most important aspect of success. If you select a column that is poorly suited to the separation, a great deal of time and money will be spent on the method development with poor results. Start with the most suitable column(s).

pH Measurement of HPLC Mobile Phase Solutions and Buffers

Several times each month I am asked how to "correctly check and adjust the pH of an HPLC buffer solution which has an organic solvent component"? Well, the answer is to always check and adjust the pH of the purely aqueous solution first. Only pure aqueous solutions can be correctly adjusted for pH in the laboratory. Do not mix any organic solvent into the water based solution until after you have correctly adjusted the pH. The addition of an organic solution will throw off the final reading. Once the aqueous portion of your solution has been correctly adjusted to the desired pH value, then you can mix the solutions (or run an organic solvent gradient against the aqueous portion) as needed.

*This procedure also serves to make sure that all solutions used in chromatography are prepared in the same manner. It is true that the pH of the final mobile phase mixture (aqueous and organic mixture) may not be the same anymore, but the prepared stock solutions from which they were made will be the same each time, insuring reproducible results. Developing and describing chromatography methods and procedures which are highly reproducible equates to good scientific technique.

Common HPLC Calculations:

Capacity Factor / Retention Factor / Capacity Ratio:  k1 (K Prime)

k1 = T(R) - T(0) / T(0)
where T(R) equals the retention time of the peak in minutes and T(0) is
the retention time of an unretained peak. *For chromatography to take place, K Prime must be > 1.00 and for most modes of chromatography, should be greater than 1.5 or 2.0 for all samples !


Tailing Factor: USP: 't'

t = W(5.0)/tw/2

where tw equals the distance between peak front and T(R) at 5% of peak height units. W(5.0) equals peak width at 5% height, in minutes.


Theoretical Plates: USP and ASTM, 'N'

N = 5.54 x (T(R)/W(50))2          

Assumes width at peak half height (50)
* More info can be found at this link.


Resolution: USP, ASTM, 'R'

R = (T(R)(b)-T(R)(a)) x 2.35/(W(50)(b) + W(50)(a))/2

Assumes width at half height (50%) with peaks (a) and (b).

*Notes: Visually, "Baseline" resolution is R = 1.5. Your goal should be R = or > 2.0. ** R of 1.5 provides 99.8% separation which means you cannot accurately quantify a 0.1% impurity so develop the method to have a resolution value of at least 2.0.


Note: The appropriate formula(s) for use with your samples may depend on which of the many pharmaceutical guidelines and regulations apply in your country. Always consult the appropriate guidelines.