HPLC Mobile Phase Composition and LC-MS Electrospray Voltage

I am often asked about the importance of selecting and optimizing the LC-MS Electrospray Ionization Interface (ESI) voltage. To better understand why it is necessary to do so and how it effects the results obtained, let us review some key facts about ESI first.

• While a gas sheathed flow of volatile mobile phase is directed into the MS source, a strong positive or negative electric field (KV) is applied across the MS inlet. The effluent is atomized and evaporated to form ions (voltage polarity determines positive/negative mode).
• Too high of a capillary voltage may produce electrical arcing resulting in damage to the system (e.g. PEEK needle may melt, burn and/or clog).
• Too low of a capillary voltage and ion evaporation will not occur.
• The voltage needed to produce efficient desolvation and ion evaporation are directly related to the sheath gas flow rate, the mobile phase composition and the flow rate.

What Can You Do To Insure Finding A Suitable ESI Capillary Voltage?

1. High quality HPLC methods which utilize fully volatile mobile phases and first retain, hold, then elute all samples are needed to generate LC-MS or LC/MS-MS methods. Optimize the HPLC column type, dimensions, MS compatible mobile phase composition and flow rate before optimizing the MS settings. If you have enough sample available, use an infusion method (continuous flow injection) to establish the initial MS settings needed to detect the sample before continuing with the LC/MS method development optimization. Infusion (with a syringe pump) provides the needed time to makes changes, observe how they change the signal for fastest optimization.
2. The HPLC mobile phase and any dissolved additives or buffers used for LC/MS analysis must be of high purity and fully volatile.
3. Make sure your sample is fully dissolved in the mobile phase and filtered (0.22 u filter) before injecting into the system.
4. Basic samples can be protonated to form [M+H]+ clusters in acidic mobile phases.
5. Acidic samples can be deprotonated to form [M-H]- clusters in basic mobile phases.
6. The electrospray ionization (ESI) process used in LC/MS or LC/MS-MS analysis is affected by the surface tension of the HPLC mobile phase used. Water has a higher surface tension than most organic solvents (i.e. Methanol, Acetonitrile, Ethanol, IPA). Using conventional flow rates with highly aqueous mobile phases requires a higher initial voltage for ion evaporation to occur. IOW: Mobile phase mixtures high in water content will require a higher capillary voltage.
7. Higher organic solvent content usually leads to better atomization / droplet formation and require less capillary voltage to maintain.
8. Lower HPLC flow rates usually lead to better atomization / droplet formation and require less capillary voltage to maintain.
9. To optimize the ESI capillary voltage it is necessary to carry out experiments trying different voltages and monitoring the signal (S/N of a standard or sample) to find the best voltage which results in good signal quality and low noise. This process requires experience to know which settings are likely to enhance the signal and a great deal of skill operating the Mass Spectrometer.

Optionally, ESI signal output may be enhanced using: Adducts or changing the solution chemistry with other mobile phase additives.

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.

Common pKa Values for ACIDS & BASES used in HPLC and LC/MS Method Development

pKa (25°C)                              ACID

0.3                                           Trifluoroacetic acid

2.15                                          Phosphoric acid (pK#1)

3.13                                          Citric acid (pK#1)

3.75                                          Formic acid

4.76                                          Acetic acid

4.76                                          Citric acid (pK#2)

4.86                                          Propionic acid

6.35                                          Carbonic acid (pK#1)

6.40                                          Citric acid (pK#3)

7.20                                          Phosphoric acid (pK#2)

8.06                                          Tris

9.23                                          Boric acid

9.25                                          Ammonia

9.78                                          Glycine (pK#2)

10.33                                        Carbonic acid (pK#2)

10.72                                        Triethylamine

11.27                                        Pyrrolidine

12.33                                        Phosphoric acid (pK#3)

Notes: (1) This is a general list of commonly used acids & bases for chromatography applications and not meant to be a comprehensive list of all values. (2) TFA is an overused and very strong acid for many chromatography applications. It also has strong ion pairing properties and can result in high UV noise, vacuum degasser and/or MS contamination. If you must use it, try and use the lowest concentration which results in the desired pH. Example: 0.1 % TFA ~ pH 2.0, 0.02% TFA ~ pH 2.7. (3) Formic acid is a popular alternative to TFA for many applications, esp LC/MS. (4) Not all acids/bases provide "buffering" on their own.

Reference: CRC Handbook of Chemistry & Physics.

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 ]

Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems

"Carry-over" is a term used to describe a type of sample contamination which causes sample peaks to re-appear in later runs which do not actually contain the sample (e.g. blank runs). The contamination can last for several sequential runs, often decreasing in amount after each injection (which is a key observation when troubleshooting). When proper instrument training has been provided, modern HPLC system designs make carryover extremely rare, but when it does appear, the contamination can be due to: (1) A lack of HPLC maintenance; (2) Overloading samples which foul the column; (3) Poor Wash Vial Usage and/or Sample Vial Selection; (4) Inadequate operator training in how to set-up and use the chromatography system. *Note: Proper operator training greatly reduces the chances of contamination and is the most overlooked reason for the problem.

The Types of HPLC Carry-Over Contamination; Why They Occur and How To Reduce Them:

(1) A Lack of HPLC Maintenance: Most auto-injector valves rely on a rotary seal to move the sample from the needle loop to the flow path of the system. The components within these valves wear out and should be inspected at least every 6 months and replaced when needed. Also, always check the needle seat and needle for signs of wear or leaking. Note: Look for signs of leaks by the injector. Leaks always indicate a problem and should be fixed immediately. Don't run samples when you have leaks. Your method and data obtained will be invalid. Any worn parts should be replaced and the system performance tested. One of the most common causes of carry-over is due to a worn sample injector valve rotary seal. A worn seal can allow sample to be retained in the worn grooves, in-between injections, resulting in sample peaks appearing in subsequent runs. Additionally, buffer salts can lodge between the seals causing leaks or carryover. Routine HPLC service and, if applicable, flushing of all buffers/salts every day can eliminate these issues.

(2) Column Fouling / Overloading of Sample: If you inject too high a concentration of sample and overload your column with material, then it is possible that your column will continue to bleed sample long after the analysis is over. This also happens when the sample has a high affinity for the support you have chosen too. Failure to regularly flush and clean your HPLC column on a regular basis can also result in a similar problem as retained material is released from the column over time. Avoid this problem by performing a loading study to determine how much material can be effectively loaded on to the column. Next, create a wash method which utilizes a stronger solvent than your method (often utilizing a gradient) which will wash away any strongly retained material in between runs. This is critical if you are running an isocratic method as material will be retained on the column and must be washed off at frequent intervals using a stronger wash solution. *When using only isocratic methods, people often do not initially observe carry-over problems (because the sample just sticks to the column and accumulates over time). When the solvent strength is changed or the method is revised to a gradient, then the problems start... Avoid the problem by selecting the right column (which retains, then elutes ALL of the sample), not overloading the column (do a loading study) and washing the column down with a stronger solution that fully dissolves (not precipitates out) any remaining material off the column after each run.

(3) Wash Vial Usage and/or Sample Vial Selection: If you are using a modern high-pressure, "Flow-Through" design autoinjector (e.g. Agilent 1100, 1200, 1260, 1290), then carryover is rarely an issue as these modern injectors use a high pressure pump to aspirate and inject the samples directly into the flow path, reducing the need for any wash stage. The entire HPLC's injection flow path is continuously washed with mobile phase during the analysis run. This dramatically reduces the chances of any sample re-appearing in later runs. The need for a separate wash vial is nearly eliminated in this way as the needle, needle seat, loop, injector pump and valve are all flushed clean during each method. Many older auto-injector designs utilize either a low pressure injector (glass syringe) or injector pump which is not part of the main flow path. These injectors benefit from a separate wash vial as they are not continuously cleaned. Effective cleaning requires that a wash vial be employed in these cases. It should be filled with mobile phase or a solution which will dissolve any remaining material which might still be in the system.

When sticky sample solutions are used, separate Wash Vials can be used to reduce contamination with either older or newer injector designs . Sometimes these sticky samples can adhere to the outside of the needle while it is being withdrawn from a vial which has a septa which has been punctured many times. High puncture rates tend to open up the hole resulting in a lack of "wiping' of the needle surface upon withdrawal. *For vials that are punctured many times, it is critical to replace the septa OR use septa materials which seal for a long enough time frame to reduce this effect. Septa needle wiping eliminates some of this contamination. Two types of contamination can occur from this problem. (a) When the needle is dipped into a vial (same or different one) which also has a large septa opening, it can carry some of the sample with it and deposit it into the new vial (or onto the septa of the vial). (b) The contamination can also run down the needle itself and drip onto the needle seat at the time of injection resulting in contamination of the seat or sample.

One of the easiest solutions to reduce external needle contamination involves incorporating a wash vial which contains a solution which is optimized to quickly dissolve the sample into solution. This sounds simple, but many chromatographer's choose wash solutions which do not enhance the cleaning aspect of the needle at all. For example: Mobile phase, which is normally ideal, but does not work in some cases. Samples such as peptides, proteins, fats, oils and/or lipids can be troublesome as their solubility can be at odds with the mobile phase chosen. For the wash vial to be effective, it must quickly dissolve the material. The needle can be first "dunked" (dipped) into one vial containing the solution and withdrawn, followed by an aspiration and wash in a second wash vial. If needed, you take this cleaning one step further and use additional aspiration steps to serially dilute any remaining material. These wash vials must be changed frequently (easily done by having several wash vial positions programmed in the system). Additionally, the caps should be left OFF the wash vials to reduce pickup contamination each time they are used (this step is critical).

Lastly, if you are analyzing sticky materials which are known to interact with metals found in chromatography systems, consider using a system which incorporates bio-compatible materials such as titanium, tantalum and/or polymers such as PEAK. You can also utilize plastic sample vials (e.g. PP) or plastic vial inserts too.

(4) Inadequate Operator Training: Good chromatography requires a complete understanding of the hardware used and the fundamentals of HPLC. You must be able to troubleshoot the complete flow path of the system and understand the concepts of chromatography as used in method development. This is not a technique best learned by trial and error, but rather through mentoring using logical steps. Depending on your skill set, troubleshooting a "carry-over" problem in an HPLC system can take minutes to months to diagnose and solve. We learn these skills through hands-on experience and training. Reading many of the better books and articles on the subject matter helps too. Get as much practical hands-on training as you can. Ask your supervisor or manager(s) to invest in you by purchasing professional training for you in this field so you can learn on your own systems. You will learn far faster this way and spend less time troubleshooting problems and more time running samples, accurately in less overall time. Training also costs just a fraction of what the instrumentation and your salary are. If you have acquired the fundamental skills, a skilled teacher can impart about one years worth of practical knowledge to you in as little as one week of training.

Summary: The two most common reasons for sample carry-over contamination in an HPLC or LC/MS system are due to: lack of operator training and/or lack of system maintenance (most commonly manifested as a worn injector rotor seal).

 Note: This article specifically addresses carry-over contamination as it relates to the most commonly used HPLC, UHPLC and LC-MS autoinjectors (or autosampler modules).

You may wish to read a related article on "Troubleshooting HPLC Injectors (Manual and Automated)" found at this link: http://hplctips.blogspot.com/2013/06/troubleshooting-hplc-injectors-manual.html

Syringe Filter Selection for HPLC or LC/MS samples

This article will address the use of disposable female, Luer-compatible, syringe filters without built-in pre-filters for the filtration of individual samples into vials for HPLC or LC/MS analysis. - Note: 96 or 384 multi-well filtering plates provide for a better solution when large quantities of samples need to be filtered. Note: The presented filter membrane material selection criteria also applies to mobile phase filtration too.

The choice of syringe filter depends on the: filter size (volume) of your sample, the chemical compatibility of the housing and membrane and desired pore size. Selection of the wrong filter size can result in too much sample holdup volume (loss of sample on filter) or overloading of the filter (allowing unfiltered material to pass through). If a membrane or housing is chosen which is not chemically compatible with your solution, then contamination of the sample or rupture of the assembly can result. Choosing a filter with too large a pore size can result in material passing through it which could clog or contaminate the solution (i.e. plug an HPLC system or result in a loss of sterility of a solution). Protein binding affinity is another characteristic of filter membranes and if you are filtering samples of biological interest, then you will also want to consider this specification in your selection criteria too (though it will not be discussed in this article).

Syringe Filter Size:

Filters are available in a variety of sizes which are generally in a disc shape and described by their diameter. Common sizes available for chromatography samples include: 3 mm, 4 mm, 13 mm and 25 mm (~25 - 30 mm) diameter discs. The larger the diameter of the disc, the larger the sample capacity, cross sectional surface area and potential hold-up volume of the sample on the filter. 

Hold-up volume is important because some of the sample will be retained inside the membrane and/or filter housing. If too large a filter is selected, samples with small volumes could be lost entirely in the hold-up volume on the membrane. Smaller filters have lower hold-up volumes. To extract as much sample as possible, be sure and use a post-filtration air purge to reduce the total hold-up volume.

If the volume of the sample you wish to filter is under 1 ml, then a 3 mm filter may provide the lowest hold-up volume and require the smallest amount of solution. To filter samples between 1 ml and 10 ml, the 13 mm diameter filter provides a good balance between hold-up volume and large filter surface area. Larger sample volumes from 5 ml to 50 ml are often filtered through the more common 25 mm diameter filters (~4 times the filtration area as a 13 mm disc).

Chemical Compatibility:

Membrane Material: This is where you really must consult the manufacturer’s own documentation for the most compatible filter membrane for both your sample and the solution that will flow through the filter. To simplify the selection criteria, we can make some generalizations about some of the different types available:

Cellulose Acetate (CA): Use with aqueous solutions and a few hydrocarbons only. Low protein binding so good for many biological samples. Not compatible with ACN or DMSO. Can be autoclaved.

Nylon: Great general purpose material and compatible with many HPLC solvents (including THF, alcohols, ACN), but not strong acids. Nylon has a high affinity to bind proteins. Can be autoclaved.

Polysulfone / Polyethersulfone Variants (PS / PES): Commonly used with tissue culture and ion chromatography samples. Stable with many strong bases and alcohols, but few HPLC solvents (as it is hydrophilic). Low backpressure and low protein binding. Not compatible with ACN. Can be autoclaved.

Polypropylene (PP): General purpose hydrophilic material with resistance to most acids, bases, DMF, DMSO and alcohols. Not recommended for use with hydrocarbons, esters or solvents such as ACN. Can be autoclaved.

Polyvinylidene difluoride (PVDF): Hydrophilic material with broad compatibility. Often a good choice for use with alcohols, hydrocarbons, biomolecules, ether and ACN. Low protein binding. Can be autoclaved.

Polytetrafluoroethylene (PTFE): Reported in most brochures to be chemically resistant to almost all solvents, strong acids and bases. Hydrophobic membrane should be pre-wetted when used with aqueous solutions. Low protein binding and very strong. Can be autoclaved.

Most chromatography grade syringe filters are constructed of either HDPE or PP. These materials are compatible with a wide range of HPLC solvents and both offer low levels of extractables. HDPE has been reported to be more chemically compatible with aqueous basic solutions of NH4OH than PP.

Pore Size:

This will depend on your application and a number of different pore sizes are commonly available from vendors (1 micron, 0.8, 0.45 and 0.22 micron are the most common): 

For example, is sterilization of the fluid the goal? If so, a 0.22 micron filter is generally accepted as the best choice.  


For most chromatography or LC-MS applications either a 0.45 or 0.22 micron filters are preferred.

Summary:

• Please refer to the various manufacturers data sheets to select an appropriate syringe filter with: (1) a low hold-up volume; (2) large enough size for the volume of sample; (3) which is chemically compatible with the solution and material you are going to inject through it and (4) lowest protein binding affinity (if applicable).
   
• To reduce the hold-up volume, use a post-filtration air purge to empty the filter.
   
• Minimize contamination from extractables (in the plastic) by pre-rinsing the filter membrane with some of the clean solution. This can reduce the amount of detectable extractables in your sample. PTFE based membranes have some of the lowest extractable levels so consider their use if this is an issue.
   
• If analyte binding is a concern, select one of the membranes which has the lowest binding affinity such as PVDF or PTFE.