Air Bubbles Exiting the HPLC PUMP, Reasons Why.

Reasons For Air Bubbles Exiting The HPLC Pump:

• Pump Cavitation: When the pump pressure fluctuates wildly up and down, at very low pressures, this is often due to 'pump cavitation'. It is caused by a loss of priming inside the pump (Air, instead of liquid is in the pump's flow path). The HPLC pump should be primed with fresh, degassed mobile phase (following proper procedures) to restore smooth, stable flow. Often, this can be accomplished using the pump, set to a high flow rate, to draw liquid from the bottles. In cases where the pump is not strog enough, manually priming the low pressure lines using a syringe (~ 20 mL) filled with mobile phase and opening (or disconnecting) a fitting at the pump's outlet may aid in priming the system. Note: Depending on the configuration of your HPLC system, to fully prime an HPLC pump, you may need to run 20 or more mLs of solution through EACH channel. Please keep this in mind every time you use the system and every time you prepare or change a mobile phase solution. This article on baseline/pressure fluctuations may assist you in troubleshooting.

• Loose Connections: If one or more of the low-pressure fittings (nuts and ferrules)  which secure the Teflon tubing to the pump (or vacuum degasser) are damaged or loose, air may enter the system resulting in bubbles. Most pumps use plastic finger-tight style fittings 1/4-28 (or 5/16-24). The threads are soft and can be deformed. When access to these fittings is difficult, sometimes the fittings are left loose and will allow small amounts of air to be drawn in. A build up of salts and/or buffers on the exposed fittings can also allow air into the system (and the presence of deposits on the fittings indicates poor maintenance and a LEAK !). Inspect the tubing and fittings used for proper type, seating depth, wear/condition, cleanliness and/or damage. Replace parts as needed and re-install using the correct amount of torque.

• Flow Rate Too High, Too Low or Not Enough Degasser Equilibration Time: Degassing efficiency is directly related to the flow rate. Lower flow rates increase the residence time of the mobile phase in the degassing membrane or tubing, improving the gas removal. Higher flow rates provide less time for gas extraction and result in lower degassing efficiency (which equals bubbles in the outlet line). Check with the manufacturer regarding the optimal flow rate range for your degasser to insure you are working  within an acceptable range. Allow enough time for the degasser to reach its set-point and stabilize before use. If the degasser is not operating properly or is unable to "keep up" with the flow rate, then bubbles may be frequently observed in the outlet lines. 

• Choice of Mobile Phase Liquid: The miscibility of the liquid is also important. If the new mobile phase is not compatible with the previously used mobile phase, pump cavitation may result. Always flush the pump with an intermediate liquid that will dissolve in both the old and new fluids to flush them out before introducing the new mobile phase solution. (such as pure water or IPA, as applicable). The solubility of air (gas) in the specific solution used also affects the efficiency of the vacuum degasser. Aqueous solutions usually hold less gas than popular organic solvents (though air bubbles can be harder to "push" through in water). The amount of dissolved gas inside the liquid relates directly to the time needed to reduce it to acceptable levels for use in HPLC. Be sure to allow enough time to properly degass the new solution.

• Dirty or Obstructed Solvent Pickup Filters (Bottle filters): Bottle filters should be cleaned or replaced at regular intervals, following routine maintenance SOPs. When they become fouled or obstructed, a vacuum may form as the liquid is drawn into the system. This may result in air being sucked into the tubing or through a fitting (remember that the low pressure Teflon tubing used to connect the bottles to the degasser and pump is porous and allows gas to diffuse through it). The pickup filters should not obstruct the normal flow of solvent (typically they are 10-20 u in porosity). * a quick troubleshooting tip to rule out an obstructed solvent pickup filter is to temporarily remove the filter from the bottle. Observe the back-pressure on the pump to see if it increases and priming is restored. If so, the filter may be clogged. Always replace the filter with a fresh, clean filter and never operate the HPLC without the solvent filters installed.

• A Sticking Check Valve: The pump's inlet and outlet check valves must function perfectly, all of the time, to maintain proper flow and pump function. If an inlet check valve is not fully closing properly to seal off the high pressures generated inside the pump, then the pump will not be able to maintain pressure or flow. Inspect the check valve. Remove and clean it, per the manufacturer's guidelines (often this involves placing the check valve assembly in a beaker with solvent such as IPA and sonicating for 20 minutes to remove any residues. If cleaning fails to restore proper valve function, then replace the check valve with a new one.

• Worn Pump Piston Seals (or Pistons): When the piston seals begin to leak, air is allowed into the system. Pump piston seals require regular replacement (they are normal wear items). Scratched or worn pistons may also result in leaks with air getting into the system. Inspect and Test them both for pressure tightness on a scheduled basis or anytime you suspect a problem. Flush the pump with a suitable liquid, then run a high-pressure test to determine if they pass or fail the manufacturer's leak tightness and high pressure tests. Be sure to perform a physical inspection too.

• Contaminated or Obstructed Pump Outlet Filter: Most HPLC pumps have a small disposable outlet filter installed at or near the pump outlet line (Note: In the case of most Agilent brand HPLC pumps, a small PTFE filter may be found at the outlet valve or inside of the prime-purge valve). These filters should be replaced at regular intervals (monthly is strongly recommended), especially if any aqueous buffers or solutions are used (a they contribute to contamination). Contaminated pump outlet filters may result in a number of pressure instability problems. Abnormally high back-pressure during operation OR when vented to waste are indications it is obstructed. Regular scheduled replacement is the best way to prevent lost time and reduce system contamination.

 Any of the above causes may contribute to valves not functioning properly or air being drawn into the HPLC system. Troubleshooting should begin with the easiest and obvious areas first. Check the condition of the low pressure tubing used to make the connections to and from the mobile phase bottles and pump. If it is kinked, twisted or damaged, replace it with new tubing. Check the fittings used (nuts and ferrules) for tightness and to insure they have been installed properly and are not leaking. Repair all leaks. Keep the system clean (it is easier to monitor and troubleshoot problems when it is clean). Replace any damaged fittings with new ones. Check the solvent pickup filters monthly to insure they are clean and not obstructed. Make sure the flow rate you are using is within the acceptable range for your degasser. 

Has your degasser module been professionally cleaned and serviced within the last 5 years? Are any degasser errors being generated? Is the vacuum degasser making any unusual sounds? Is liquid being emitted from the vacuum pump exhaust port? If any of the answers to these questions are 'yes', then have the HPLC vacuum degasser professionally diagnosed for problems so that repairs can be made to restore function.

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

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

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

As a general guideline, we shall not place our mobile phase reservoir bottles in any type of dishwasher or wash them using any dish soaps. These may leave a residue easily detected by even the weakest mass spectrometer. Avoid contamination by purchasing high quality glass bottles with vented caps to keep dust out. If rinsing with organic solvents (and/or freshly prepared and filtered high resistance water) does not clean them, you can try a Nitric Acid rinse (up to 30%) followed by a neutralizing wash in 2M Sodium hydroxide. Follow-up with many rinses of HPLC Grade water (or LC/MS grade), oven drying, then re-fill with an appropriate mobile phase. Don't forget to replace those solvent pickup filters too. While many 316 SS pickup filters can be cleaned, most of the sintered glass style filters are designed to be disposed of (not cleaned or put in an ultrasonic cleaner!). So periodically dispose of the glass types and install new filters and fresh mobile phase into those recently cleaned bottles (before you start looking for the source of contamination in the more expensive parts of the instrument, clean or replace the filters). - Please don't re-contaminate an expensive HPLC or LC/MS system and invalidate your methods and data because you skipped replacing a $10 part. Keep commonly used spare parts in-stock and always maintain a clean system.

HPLC Peak Splitting. Common Reasons For It

True "Split" HPLC peaks, not resulting from co-elution of another peak, can be caused by a number of chromatography problems. Here are a few examples and their solutions:

1. Sample overload. Sample overloading is one of the most common reasons for observing peak "splitting". Reduce the sample concentration by factors of ten to see if the peak shape improves. 
2.  A poor quality HPLC method. Poor quality methods which do not use mobile phase solutions which are at an appropriate pH (*If the pH of the mobile phase is close to the pKa of the sample, then split peaks may result); which does not dissolve the sample in (should be fully soluble) or are unstable, show sample or mobile phase precipitation can cause this effect. Always check solubility before starting.
3. A partially plugged or fouled column. A dirty or fouled column (from not washing down properly with a solution which is STRONGER than the mobile phase). Analysis methods should be followed by separate wash methods to remove all bound material and any late eluters,
4. Wrong injection solution. Peak splitting may be the result of dissolving and injecting your sample in a solution that is stronger than your mobile phase. Dissolve and inject samples in the mobile phase or in a solution which is a slightly weaker solution (not stronger).
5. A poorly packed column, void at column inlet, a dirty frit or poor mechanical connection (i.e. improperly swaged fitting). These types of structural or mechanical defects can each result in peak "splitting" (all of these are less common today than in the past using modern HPLC columns). When present, a dirty inlet frit can be replaced with a new one, or the column can sometimes be backflushed to remove any accumulated material. Connections should always be double checked.
6. Detector data rate set too low. Too few peaks collected over time may result in integration errors and inaccurate peak symmetry problems. Read more about how to determine the best data collection rate at this link.

An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?

The introduction of electronic vacuum degassing / degasser modules to the liquid chromatography industry a few decades ago has introduced several new problems which were unknown years ago when we sparged our mobile phase solutions with high-purity helium gas. One of these problems relates to how the electronic vacuum degassing modules themselves can contribute to contaminating your HPLC or LC-MS system.

Before using an HPLC vacuum degasser module, please review all of the information and advice supplied by the manufacturer of your specific degassing module. The composition of the internal degassing tubing has changed a great deal over the past decades resulting in increased degassing efficiency, but also changes in mobile phase chemical compatibility. Some popular solvents may be incompatible with some models in your lab. Make sure you know exactly which types of vacuum chambers and plastics are used in your specific instrument(s). Degassing modules must be operated, cleaned and maintained the same as your other important instruments. When they are not operating properly and/or are contaminated, they should be serviced as soon as possible or risk further contamination and damage to your system ($$$).

•  Link to a pdf document with pictures showing common contamination and damage observed while inspecting HPLC Vacuum degasser modules for our clients. Have you had your HPLC vacuum degasser(s) professionally evaluated for damage? [ https://www.chiralizer.com/examples_of_degasser_contamination.pdf ].
  

In a previous post ["Inline HPLC Degassing Modules"] we discussed the convenience that these devices have brought to our laboratories, but also the extra training requirements (such as cleaning and flushing the vacuum channels every day and routine servicing every 2 to 5 years) which must be undertaken to use them successfully. When the operational guidelines for the use of these products are ignored, these devices can contribute to the contamination of your HPLC and /or LC-MS system. The internal wettable surfaces of each degassing chamber contain plastics (examples of plastic used: Teflon, Teflon AF, Tefzel and/or Peek are the most common types of used). To effectively remove gas from the mobile phase, the liquid must pass through plastic tubing (or across membranes) placed in a vacuum, for a period of time which is long enough to allow a portion of the dissolved gas in the mobile phase to diffuse through the degassing tubing/membrane and out the exhaust port of the degassing chamber. The degassing tubing (most use tubing) should have the maximum chemical compatibility possible while allowing it to also be porous enough for the gas alone to diffuse through the walls of the tubing under vacuum. These requirements usually result in some type of fluoropolymer tubing (Variants of Teflon or Teflon AF) being used as they have broad chemical compatibility plus can be formed with controlled pore sizes for the effective removal of gas, not liquid, through the tubing walls. However, there are exceptions to this and the plastic(s) used may NOT be chemically compatible with all liquids used in chromatography applications. Depending on the plastic degassing tubing used, the tubing may swell, fail or even dissolve into the mobile phase solution! Be sure and check the chemical compatibility chart offered by the degassing module manufacturer for compatibility with your mobile phase and ALL additives used before using the instrument. Some examples of incompatible chemicals on the lists of many instrument vendors include: THF, Chloroform, DCM, strong acids or bases, Hexanes and Sodium Azide. Use of incompatible solvents or additives may result in complete failure of the degasser module plus contamination of the entire instrument flow path. We have seen many degasser systems which were used with (or stored in, w/o proper flushing) strong acids show corrosion of the metal parts inside the chambers (SS fittings and connectors) forming piles of rust and salts which were carried through the vacuum system resulting in damage to the system and flow path contamination. *Please do not risk it. Be aware of which chemicals may pose a risk with your system. For example: The use of many fluorinated solvents may dissolve most types of tubing when Teflon AF is used for degassing.

• Note: We have seen an increase in the use of various perfluorinated solvents, esp with LC/MS systems. This has resulted in severe degasser damage plus MS source contamination (e.g. HFIP and Ethoxynonafluorobutane). Most perfluorinated solvents are not compatible with vacuum chambers which contain Teflon AF. They may dissolve the degassing tubing, resulting in the destruction of the degasser chambers and contamination of the vacuum system and mobile phase (IOW: the complete HPLC system flow path). Additionally, we commonly see ion-pairing reagents such as TFA and TBA "sticking" to the plastic used in these modules causing a leaching of material over long periods of time (again, most obvious on an MS system where you can "detect" it in the background signal). These ion-pairing agents must be thoroughly flushed out of the flow path to reduce contaminating the entire system over time. *A strong wash solution with a little acid (formic) alternated with a wash containing some base (ammonium) often helps in this regard. Wash cycles of over 12 hours are often needed to remove these compounds and see improvement (It may take much longer...). In some cases we must replace some or all of the internal parts of the degassing module to eliminate the contamination. Always remove any HPLC column from the flow path (to avoid re-contaminating again) and replace with a new one, once the contamination has been removed. It is for this reason that we should avoid the use of strong ion-pairing reagents in any LC/MS system, as they often contribute to very high background signals and long term contamination. *Helium sparging should be considered for such applications.

Reversed phase HPLC applications which use highly aqueous mobile phases may under some circumstances result in high rates of pervaporation of the water vapor into the degasser module resulting in condensation of the water into the vacuum system (Unlike the older Teflon material used, the newer Teflon AF formula is more permeable to water vapor). Once liquid enters the vacuum pump, severe damage has already occurred and failure of the vacuum system soon follows. *If you ever notice liquid exiting through the vac pump's exhaust port, turn off the HPLC system and have the degasser module professionally serviced. 

Another common problem seen when aqueous solutions are used in an in-line vacuum degasser are that of algae and bacterial growth. Most often observed in systems left unused for a period of time or which are not periodically flushed out with organic solvents. Growth inside the low pressure tubing and even inside the vacuum chambers has been known to contaminate the entire flow path of an HPLC system. Replacement of the tubing and internal chambers usually resolves the problem.

Buffers / Additives: Just as with the rest of your HPLC system, any buffer salts, acid, bases or additives which are left in the system (even overnight) can damage it. This is true of the vacuum degasser module too. Please be sure to flush all of the vacuum degassing chambers of any salts or buffers when not in use.

For normal phase applications, high concentrations of n-Hexane may cause contamination or damage to a degasser attached to an HPLC or LC/MS system. Some types of degassers are not compatible with Hexane. The ultra high evaporation rate of hexane coupled to the advanced materials found in the degassing tubing or membranes may result in the hexane condensing on the outside of the internal degassing tubing of the degasser and then aspirated into the vacuum system (causing damage). The contaminants are then transported back through the tubing walls into the solvent stream (your mobile phase).

If your HPLC's vacuum degasser fails to achieve vacuum, has liquid exiting the vacuum pump exhaust port (or exhaust tubing) or shows an error (e.g. Leak Error, High RPM, makes loud noise, a Yellow or Red light on an HP/Agilent system or "Degasser Hardware Fault" / "Degasser High Leak Rate" messages often seen on Waters brand systems), then your entire HPLC system may be out-of-compliance - because the degasser is broken. Have the vacuum degasser professionally cleaned and repaired so you can put the system back online. Do not assume that only the vacuum pump has failed, as replacement of the pump alone often results in failure of the replacement pump soon after (due to contamination and other problems incorrectly diagnosed). The true cause of the failure must be correctly diagnosed and repaired first, and this is something best left to professionals.

Our professional HPLC degasser repair shop receives many types of degasser modules with leaking or ruptured vacuum chambers. These directly contribute to mobile phase contamination as any seal failure in these normally "dry" systems results in liquid contaminating the vacuum system which in-turn sends contaminated liquid and vapors back into the HPLC mobile phase stream. *Note: If you are using your HPLC degasser with Mass Spec detector, then the resulting mobile phase contamination may contaminate not just your column, but MS source too (costing a great deal of money to decontaminate). Please, at the first sign of trouble, have the degasser professionally diagnosed, cleaned and repaired. For more information on having your degasser professionally diagnosed and repaired with fast turnaround at a fraction of the price charged by most instrument vendors, please refer to this link: "HPLC Degasser Repair Service" [ http://www.chiralizer.com/hplc-degasser-repair.html ]

Gradient Mixing Test For Your HPLC Pump (Step Gradient)

The most popular type of gradient pumping module used to perform HPLC analysis utilizes a low pressure mixing valve in their design. These valves are electronically controlled and proportion the amount of mobile phase from one of several solvent channels into a mixer for introduction to the pump head (*the solenoid valves used for this are sometimes called gradient proportioning valves). They provide random access to multiple solvents (e.g. 4) for method development and column flushing. The mobile phase solutions are mixed at low pressure before entering the high pressure side of the pump head (where they undergo compression). This design requires only one high pressure pumping head and can allow for very high mixing accuracy (often 0.1% per channel) of the mobile phase. This allows for the formation of mobile phase gradients over time which greatly aid in resolving samples apart on the column.

The gradient proportioning valves need to be tested along with the other parts of your HPLC system on a regular basis to insure they are operating within the manufacturer's specifications. They should also be tested anytime you suspect a problem may be present. One quick way to check the operation of two of the valves is to use a tracer compound and STEP gradient to monitor their operation. You can set up a method to perform this test as suggested below.

QUICK GRADIENT COMPOSITION TEST:

Bottle A = 100% DH20;
Bottle B = 0.1 % Acetone in DH20 (*Acetone is the tracer compound);

Flow Rate = 1.000 ml/min;
Column = No column. Install a restriction capillary in place of the column to obtain a backpressure of > 60 Bars;

Detection = 265nm (10 nm bandwidth) UV;

STEP Gradient Program:
    0 to 2.00 min, 0 % B
    2.01 min, 20% B
    4.01 min, 40% B
    6.01 min, 60% B
    8.01 min, 80% B
  10.01 min 100 % B
  12.01 min 20% B
  14.00 min 20% B

Note: If the delay volume (dwell volume) of your system is large, then you may want to adjust the time values shown to LARGER values (i.e. 2 minutes delays are used in this example, but 5 or even 10 minute delays between steps may be more appropriate if your system has > 1 ml dwell volume.

Running the above method should result in a signal trace which shows a step-wise rise to 12.00 minutes (as the acetone concentration increases). The edges of the "steps" should be sharp and the risers should also be close to vertical. The final step change which starts at 10.01 minutes shows a linear gradient change back down to the 20% B level. This line should not have any bumps or dips in it and should transition smoothly back down. The height of the baseline at this point should match the height seen between 2.01 and 4.00 minutes (same 20% B). The height of the proportional steps (e.g. 20, 40, 60, 80) should also be the same. You can use your CDS to measure these height values.

Another useful aspect to view is the S/N ratio at each step. Use your CDS to establish noise windows within each range (e.g. 2.50 to 3.5 minutes). This data is useful when comparing the performance of the pump at different intervals.

If you observe deviations in the height of the proportional steps or dips in the lines, these can be caused by leaking or sticking check valves as well as leaking or sticking gradient proportioning valves. *If you have a quaternary pump, be sure and test all four of the valves used (2x per test).

Lastly, the above example is a generalized method and may or may not be applicable to your specific HPLC pump. Be sure and customize a test method which takes into account the pressure ranges, flow rates, delay volume, mixing volume, and number of low pressure channels used in your pump.

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.

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.

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 Solvent Properties Table (e.g. Polarity, Density, Boiling Point...)

Here is a link to a table listing common chromatography solvents and their physical properties (e.g. viscosity, polarity, UV absorption...). 

http://www.hplctools.com/lcsolvent.htm

HPLC PUMP SOLVENT COMPRESSIBILITY VALUES

Have you ever noticed excessive pump ripple (baseline noise) that is not caused by a defective check valve ? The ripple might be due to an incorrect HPLC Pump solvent compressibility setting.

We normally think of liquids as not being compressible in general. Hydraulic systems take advantage of this physical fact and many innovations have been developed using this concept. However, in high pressure liquid chromatography (HPLC) we routinely subject different liquids to very high pressures which can result in measurable liquid compression. The degree of actual compression varies for each liquid (see table). Though the amount of compression is very small, it is enough to change the flow rate of the system. When multiple solvents are mixed together at different proportions, such as is common when running a gradient, the measured flow rate can vary from the set flow rate during the entire run. This flow rate accuracy issue can be compensated for using the built-in solvent compressibility compensation software which is found in most modern HPLC systems. Many of these systems will allow you to manually enter the actual liquid compressibility values for each solvent (pump channel) used. This can result in better baseline stability and less pump noise. I would like to point out that the small improvement gained in performance is best implemented AFTER other major changes have been addressed first (i.e. such as fully degassing your solvents; filtering samples before injecting; selecting the best signal bandwidth and sampling rate values for your detector and insuring that your pumping system has received regular maintenance). 
 
Note how Water has a compressibility value of ~ 46, but a very common solvent such as Methanol has a value of 120. These two are very different. *Most pumps are pre-set with a compressibility value of '100'. A 50/50 mixture of the two run isocratically might benefit from a manually edited compressibility value of 83 [(46 + 120) = 166 / 2 = 83)]. *This is a best guess value as the best compressibility value for a mixture of liquids must be determined through actual experiments. Choose the value which results in the lowest pump pressure ripple and/or noise. 

SOLVENT COMPRESSIBILITY VALUES TABLE:

Solvent	Compressibility (10-6 per bar)
Water	46
Acetone	126
Acetonitrile	96
Benzene	95
Carbon Tetrachloride	106
Chloroform	100
Cyclohexane	113
Dichloromethane	99
Ethanol	112
Ethyl Acetate	113
Heptane	144
Hexane	158
Isopropanol	100
Methanol	120
Tetrahydrofuran	97
Toluene	90

Notes: 
(1) The values shown above are approximate and assumed to be accurate. They were recorded at a temperature of 20C (Reference: Handbook of Chemistry and Physics #90). Various grades/purity of solvent may have different compressibility values so please verify the values of your own solvents before use. These should serve as a general guideline only.

(2) The variation in pressure which occurs between the pump piston compression and decompression strokes are sometimes reported by the pump's electronics to aid in troubleshooting. Agilent/HP brand systems refer to it as the pressure "ripple" (should be less than 0.5 %) and Waters brand systems report the calculated ratio, "Compression / Decompression Ratio" value using this guideline [1.0 - 1.4 = Normal; 1.4 -1.8 = Fair; > 1.8 = Possible Bubble]. In all cases, continously degass all liquids and input the correct compressibility values for each mobile phase solution to achieve the most stable flow.