HPLC Solution Degassing, Sparging (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.


Sometimes 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 choice of gas matter? For liquid chromatography applications we use helium gas because it is one of the few inert gases which are NOT soluble in water and other solutions. Gases such as argon and nitrogen are soluble in most liquids. Helium displaces oxygen and nitrogen from solution quickly and easily while not adding gas to the solution. Helium is not soluble. If we sparge with argon gas, then we infuse the liquid with gas (because it is soluble in it) which is the exact opposite result we are looking for when we degas a solution. So I suggested that they replace their high pressure argon cylinder with a tank of helium. Luckily they still had their original helium tank available so we hooked it up. I sparged their mobile phase with the helium gas for about ten minutes then primed the pumps with the solution. 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. If gradient elution is used, then the gradient profile often must be changed to compensate for changes in void volume of the column and 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.

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

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 organic mobile phases. It is often described as a gentle form of chromatography leaving the protein 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.

Basic Principle: Used to separate molecules based on their molecular size in solution. 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. 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.

Support Types: Available supports are most commonly based on either silica gel or polymeric materials.

Technique: Improved resolution often results from chaining columns together with the same pore size. A broader range of molecules can often be separated using multiple columns with differing pore volumes together (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. Notes: 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 can cause damage to the pump, injector and other components if not washed 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. Salt should never be visible on the outside of the instrument.

pH 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:  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. *K Prime must be > 1.00 !


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 width at 5% in minutes.


Theoretical Plates: USP and ASTM, 'N'

N = 5.54 x (T(R)/W(50))2          

Assumes width at half height (50)


Resolution: USP and 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).

*Baseline resolution is R = 1.5. Your goal should be R > 2.0. 



Note: The correct version of each chromatography formula and/or calculation will often be determined based on the specific areas pharmaceutical guidelines (Pharmacopoeia). Many different versions of the above formulas and calculations exist and we are only presenting examples of them. Please consult the appropriate guideline for your country.

Determining the Data Acquisition Rate (Sampling Rate) For Your HPLC Detector

Another common question I am asked is how to set-up the HPLC detector’s sampling rate. This applies in a general sense to UV, VIS and many types of mass detectors (not MS). Most instrument manufacturer’s input default sampling rate values into their software packages. Often, clients use these values without questioning if they are correct. The manufacturer can not know what sample(s), method and conditions you have so you should always calculate the correct value yourself to make sure it is appropriate for your application. 

How Many Points Across The Peak Are Needed? First, some background info. In a typical chromatographic analysis you must determine the sampling rate of the detector. This value is required for proper instrument set-up, quantification and integration of your sample(s). In the most basic sense, the area under a peak requires at least ten points to describe it with some accuracy. Ten points will provide basic data about the shape of an ideal peak to the computer. Since peaks are not always perfectly symmetrical, a larger number of points will provide a more accurate picture of the peak’s shape which will also improve reproducibility and quantification. We suggest you have between twenty to thirty data points to allow for a more detailed fit to the peak. Too few points across a peak and you lose detail and sacrifice reproducibility. Too many points and you start to introduce noise into the system.

With these facts in mind we can next think about the detector’s data acquisition rate. You must select a data rate (sampling rate) that is sure to provide the recommended 20 to 30 data points across the peak width (we use the commonly calculated peak width at half height as the time measurement). Select a detector sampling rate that will provide you with this degree of detail. Take a look at your existing chromatogram and use one of the narrowest sample or standard peaks as an example to determine the best rate settings.

Examples:

(a) If your narrowest peak has a peak width of 1.00 minute (60 seconds), then divide 60 seconds into 30 parts for a result of 2 seconds per data point. The preferred sampling rate would be 2 seconds, 0.03 minutes or 0.5 Hz (depending on the units used by your detector).

(b) If your narrowest peak has a peak width of 0.20 minutes (12 seconds), then divide 12 seconds into 30 parts for a result of 0.4 seconds per data point. This equals a sampling rate of 2.5 samples per second or 2.5 Hz.

Summary:  

     To Determine the Data Acquisition Rate For Your Detector You Need To:

• Use your existing integration results to identify the narrowest chromatographic peak in your analysis.
• Record the peak width at half height for the narrowest peak (usually in units of time).
• Divide this number by thirty (30) to determine the preferred sampling rate.
• Use this value, or a value close to it, for your detector’s sampling rate.

Capillary Tubing Connection Volumes:

The length and internal diameter of the HPLC interconnecting tubing used in your system really does matter. The total volume contained in the tubing can dilute your sample or separated peaks. This can effectively undue the work of separating the peak(s) on a column. Extra volume in the tubing can also have the effect of increasing the gradient delay factor for your method (the greater the volume of the tubing from the pump head to the column inlet, the greater the delay in the solvent mixture arriving at the column). In general, keep the the total delay volume as low as possible. This is accomplished by connecting the various modules together using the shortest lengths of tubing possible. For systems which use standard sized HPLC columns (e.g. I.D.'s of 3.0 to 4.6mm and lengths from 100mm to 300mm) the tubing internal diameter should be 0.17mm (0.007"). For systems which use very short, mini or micro bore sized HPLC columns (e.g. I.D.'s of 1.0 to 2.1 mm and lengths from 50mm to 250mm) the tubing internal diameter should be 0.12mm (0.005"). Looked at another way, if the total column volume is less than 750 ul, consider using the smaller internal diameter tubing (0.17mm) to reduce band broadening. 

Here are some tubing volumes to help you evaluate the effect changing the I.D. or length has on the tubing that you use.

I.D. (mm)	I.D. (inches)		ul / cm	ul / inch
0.12	0.005		0.127	0.323
0.17	0.007		0.249	0.632
0.25	0.010		0.507	1.288
0.51	0.020		2.026	5.146
1.02	0.040		8.103	20.581

HPLC Mobile Phase Filtering & Solvent Inlet Filters

HPLC Mobile Phase Filtering: 



The tubing and valve passageways of the HPLC system are very narrow and clogs can result from using solutions which have not been properly filtered. Columns are expensive and will also clog up with particulate matter causing increased back pressure and/or changes in retention times. Running clean, particulate free HPLC grade solvents through your chromatograph is a basic maintenance requirement. High grade chromatography solvents (and ultra pure water) are often pre-filtered through 0.2 micron filters by the manufacturer to meet their grade for use in chromatographic systems. However, there are times when you also prepare (mix) your own mobile phases using theses solvents with or without chemical reagents and additives. When you prepare mobile phase using these reagent grade chemicals or additives you should also take the extra time to filter the final mixture through a 0.2 micron glass or steel filter prior to use. This helps to insure that you start with as clean a solution as possible. *This is a critical procedure to follow with buffer solutions. When using aqueous solutions, possible bacterial and algae growth can occur so remember to date the solutions and dispose of them after a suitable time period (Make up only what you will use in one week). Do not re-filter these solutions and then use them again.


HPLC Solvent Inlet Filters:

Most HPLC manufacturer's supply solvent inlet filters on the lines which draw solvent into the pump head. To protect the pump and components downstream, these lines often incorporate a filter. These solvent pre-filters are usually made from plastic (PEEK or PEAK), glass or stainless steel. Their porosity is typically ten or twenty microns. A smaller porosity could be used, but it would restrict the lines ability to draw up fresh solvent into the pump head at the required flow rate so a compromise in pore size is necessary. The filter is primarily designed to stop the pump from drawing up any large particles or debris which could cause damage to the system and is NOT used to filter the solution (as mentioned above, the solutions used should be pre-filtered). These filters can clog up over time and so should be monitored for restrictions. Stainless steel filters can be cleaned using sonication and heat. Plastic filters should usually be replaced with new ones. Glass filters, which are often made of sintered glass, can be washed, but should never be sonicated to clean them as this can cause the glass to fracture and plug them up even worse. When in doubt, replace them with new filters. Filters used with clean organic solvents often last for many years. Filters which are used with aqueous solutions last for shorter times due to build up of undesirable biological matter.

• Another way in which you can insure a clean source of liquid for your HPLC system is to make sure that your mobile phase reservoir bottles are clean and free of dirt and dust during use. Keep them covered. Always wipe off any dust and debris from the solvent bottles before you uncap them and pour them into another container (much of the dust in the mobile phase comes from dirt that falls into the bottles). Instead of 'topping-off' bottles, replace them with clean bottles containing new solution.

HPLC PUMP SEAL WASH & FLUSHING THE HPLC

Many vendors offer an HPLC Pump "Seal Wash" option. If you often operate your instrument with high concentrations of aqueous salt buffers (e.g. Protein, Peptide Separations), then an optional seal wash system might be something you want on your HPLC system. When combined with daily flushing of the HPLC system to remove buffers, it can extend the life of and reduce the maintenance needed on your HPLC system.

To prevent the build up of salt crystals inside of the narrow bore tubing, pump and other HPLC components I strongly recommend that you wash the system down each day, after use. I routinely see HPLC systems with white fluffy crystals built up around the pump heads, pistons and various fittings from lack of maintenance on a daily basis. High concentrations of mobile phase buffer in your system (e.g. 0.1 M is considered 'high', but all buffers should be flushed out) can damage the pump pistons, pump seals, injector parts and are corrosive to the stainless steel used. The resulting damage can lead to expensive repairs.

• Two types of flushing techniques can be employed to reduce the damage caused by these salt buffers and extend the life of the system. Flushing the entire HPLC flow path and optionally, flushing the back of the pump pistons using a "seal wash" system.

(1) Flushing the HPLC Flow Path: Potential damage from salts can be avoided if you remember to always flush down the entire flow path of your HPLC each day (and anytime it may sit unused) with a proper mixture of HPLC grade water and some organic (to prevent the growth of 'critters'). Flush the column down first with an appropriate solution and then remove it from the flow path. Next flush the entire HPLC system down to rinse it of any remaining deposits (sometimes the column can be left in-line and flushed with the system. Consult your column manufacturer for advice). The exact mixture to use will depend on the exact type of mobile phase you are using. You want to select something which will dissolve the buffer used in your mobile phase into the solution plus incorporate some organic solvent component to reduce the surface tension and also deter the growth of bacteria over time. For example: A common Reverse Phase (RP) solution of 80% HPLC Grade water and 20% Methanol can be used in many applications. If you have an automated HPLC system, then this entire process can be stored as a "Flush" method and programmed to run at the end of each day's sequence or series of runs so you do not have to remember to do it manually.

(2) Seal Wash System Use: A second level of flushing buffers from the system involves the use of "seal wash" pump. These pumps are often small peristaltic pumps with silicone tubing connected to them (Some are simply gravity fed systems where the wash solvent bottle must be kept elevated and waste tubing kept low to function). The tubing is connected to a metal ring which surrounds the back of the main pump's piston in such a way that it can wash liquid over it and remove these deposits. When run with buffers, the main HPLC pump pistons receive a thin coating of the solution each time they complete a full stroke. Over time, the liquid evaporate and a film of buffer salts deposit on the sapphire pistons. These salts accumulate and can scratch the pistol surface allowing air to enter the system or leaks to results. Premature replacement of the pump head seals and pistons often results from this damage. Washing the internal flow path of the HPLC system (as described in section #1 above) does not wash away these salt deposits which occur outside on the piston surface. A "seal wash" system can be employed to assist to deal with the problem. The seal wash pump's inlet line can be placed in a bottle with fresh wash solution and through either an automatic timer feature set in the pump's software or through the operator manually turning the wash pump on and off, it can wash the back of the piston to remove these deposits. The solution used to wash the pistons will again depend on the type of mobile phase you are using (just like the HPLC flushing solvent). For most RP applications, I recommend a mixture of 80% HPLC Grade water and 20% Methanol. Another common seal wash solution is 90% HPLC Grade water and 10% IPA. For most applications, I prefer using Methanol over IPA because it is much better at dissolving the buffers used. A third option would be to use a wash solvent which is the same as your mobile phase, but without any buffers added. Again, you must review your own method to determine which wash solution is best as their is no such thing as a 'universal' wash solution that can be used with all methods.

If you are running Normal Phase (NP) applications, then the seal wash can also be employed to keep the pistons 'wet' during operation and avoid excessive piston squeak noise which is common when running dry solvents (e.g. Hexane). Manufacturers often provide special piston seals designed for use with normal phase solvents, but sometime the incorporation of the mobile phase as a seal wash solvent can lubricate the pistons well. IPA can often be employed as a seal wash solvent choice too. In any case, always make sure that the silicone tubing used in your seal wash pumps is fully compatible with the wash solution you choose.

Column Temperature in HPLC / UHPLC

Let us not forget the role of temperature in liquid chromatography. Just as mobile phase composition changes are used to develop better methods, column temperature is an important chromatography variable which must be addressed. I would like to call to your attention to a few different ways temperature can change your chromatography in this "hint and tip".

(1) Stability & Reproducibility of the Method: 
Maintaining a stable column temperature during a separation is important. Excellent temperature stability can lead to a high degree of reproducibility (*Their are of course many other factors to consider as well). For a typical analysis, temperature stability of 1.0 °C / hour (over the course of the analysis) is usually enough. If you are not using a thermostatted column compartment to perform your chromatography you may have already noticed the hour-to-hour or day-to-day fluctuations which can result from running samples under ambient temperature conditions. The normal changes in room temperature can be several degrees C over an eight hour period. These types of temperatures changes can make it impossible to achieve reproducible results for some samples. It is for this reason that it is critical that you include some type of temperature control as part of your method. Always record the temperature at the start and end of each run and include this data with your report. Most of the automated chromatography data systems provide this data as standard today and it is very valuable in reproducing the data as well as for troubleshooting, if needed.

(2) Back Pressure:
Column back pressure is directly changed by temperature. As the temperature rises, the column back pressure decreases. As the temperature decreases, the back pressure increases. This can be a useful variable when working with some of the newest sub-two micron particles on the market. The very high back pressures produced by these particles can be significantly reduced by increasing the column temperature [See "Pressure Drop Across an HPLC Column" http://www.hplctools.com/Tip%20114%20Pressure%20Drop%20Across%20an%20HPLC%20Column.htm]. 

When practical, try experimenting with your method by increasing the temperature, in increments of 5°C, to measure the change. You may discover an improved method with lower back pressures, a shorter run time and sharper peaks.

(3) Viscosity: 
Viscous mobile phase systems can take advantage of using higher temperatures to reduce the overall system back pressure. Since efficiency often improves with higher temperatures a double bonus of higher efficiency (sharper peaks) and lower back pressure can be achieved just by increasing the column temperature (peaks sometimes change elution order too so use standards to check this). 

(4) Practical Considerations:
Their are limits to using higher temperatures in chromatography which must be respected. The stability and solubility of your sample, the boiling point of your solvent, the maximum temperature setting of your column heater (mobile phase, flow cell and the rest of the HPLC system) and the stability of your column over time will determine how far you can safely push this.

(5) Specifications: 
One other issue worth mentioning here is that many traditional silica columns can loose their bonded phase at temperatures above 60°C. Some specialty silica phases (i.e. Waters XBridge & Zorbax StableBond) have temperature ratings to ~ 90°C. The more exotic non-silica based supports (e.g. Zirconium, graphitized carbon and/or PSDVB) often provide poor efficiency compared to the silica based products, but can handle temperatures in excess of 100°C. 

*Always consult with the column and/or instrument manufacturer to determine what the correct and safe operating conditions are before using any instrument, column or chemical.