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.