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 ]

HPLC K Prime. Also known as: Retention Factor, Capacity Factor): One of the Single Most Important HPLC Parameters of All

The role of Capacity Factor / Ratio (K prime) in liquid chromatography is to provide a calculation or ratio which defines how much interaction the solute (sample peak) has with the stationary phase material relative to the mobile phase (IOW: the relative time the sample spends interacting with the support vs. the mobile phase). If this interaction is too short (i.e. K prime less than 1.0), then no chromatography has taken place and you have just developed a "flow-injection" method (the same as if no column was used) instead of a chromatography method. The method fails all validation and is NOT fit for purpose. Sample Retention must be long enough to demonstrate that the method developed is specific to the sample and shows good selectivity (retention) for the sample analyzed. This is true for most, but not all modes of liquid chromatography (3). 

• New and ever 'experienced' users lacking training in HPLC often make this error, developing methods where the sample has little to no retention on the column. We routinely review methods where the actual K prime of the key sample is measured to be at or near 0, failing to show any chromatography took place in the method (*The Journals are filled with thousands of examples of invalid HPLC methods of analysis). This mistake is made because the author(s) have no formal training in liquid chromatography and do not understand the basics of the technique. 
• Note: Slowing down the flow rate to "show" a later elution time DOES NOT increase K prime (a very common novice mistake) !!! 
  

Observance of the fundamentals of chromatography are key to developing high quality HPLC methods. For most modes of HPLC, highest on this list of basic fundamentals is that the sample(s) be retained on the HPLC column used and not eluted out at or near the column's void volume (we often refer to this time in minutes as, "T-zero" or "t0"). Many chromatography methods fail this simple test of retention and are invalid as written. Knowing what a compound's retention or capacity factor is allows us to be confident that it has been retained and eluted past this critical point, but to first calculate K prime, we first need to know the HPLC column's void volume. 

Calculation and/or measurement of the Column Void Volume should be one of the very first chromatography method development tasks you learn to perform. Knowing the column void volume allows you to determine the retention time of an unretained sample and the resulting retention factor (K prime) of each sample eluted after it. To do this, you must calculate the column void volume AND inject a sample which will not be retained by the column to determine what time an unretained sample will be eluted off the column. This establishes the 'T' zero time, or T(0). The time it takes an unretained compound to elute off the column is critical to know. If your HPLC method does not retain the sample on the column long enough past this time, then you are not allowing any chromatography to occur. Once you have this T(0) value, you can then determine the retention factor (the "K Prime") of your actual sample(s) using the simple ratio formula below. Your final method should baseline separate all compounds apart and, if properly developed, each sample peak will often have K Prime values between 2.0 and 10.0. K prime values of greater than 10 are acceptable, but often show minimal improvements to resolution. Try and insure that the earliest eluting peak in your sample has a K Prime of  >1.5. Do not develop methods which only result in K Primes of less than 1.5 (an indication of poor quality chromatography). 

Note 1: Many regulatory agencies (e.g. The USA FDA) requires that K prime values for HPLC separations be equal to or greater than 2.0 to meet Specificity acceptance criteria (System Suitability/Method Validation). After all, if it elutes at or near the void volume, then your method is not specific for anything. Besides being unscientific in design, your method will fail System Suitability and fail validation. IOW: It does not meet this basic requirement.

• K Prime, K1 (Capacity Factor or Retention Factor) Formula:

       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). 
• *The 'K Prime' of your sample must be > 1.00. A value greater than 1.5 should be your goal.

Example #1: 
 T(0) found to be 2.90 minutes and the sample elutes at 5.80 minutes. k1 = 5.80 - 2.90 / 2.90. k1 = 1.00.

Example #2:
 T(0) found to be 2.90 minutes and the sample elutes at 9.10 minutes. k1 = 9.10 - 2.90 / 2.90. k1 = 2.13.

Example #3:
 T(0) found to be 1.75 minutes and the sample elutes at 1.74 minutes. k1 = 0. No retention and no chromatography have taken place at all. The method is invalid.


Note 2: I see and read published HPLC methods (including "Validated Methods" !) every week which ignore this fundamental requirement and present data showing little to no retention of the primary sample on the column. Most are RP methods run on popular C18 columns and show the main peak of interest eluting out as a nice sharp peak right at the void volume.  These methods often describe the sample analyzed as "100% pure" and are fully validated (because the person doing the work may not have had any HPLC experience or training)! A mixture will always look like a single peak by HPLC when no 'chromatography' is employed to separate out all of the possible components. The sample must be retained on the column for a period of time before we can conclude anything about its purity by the method employed.

Note 3: In some cases, when other modes of chromatography are utilized (e.g. ion exchange, size exclusion chromatography (SEC / GPC), K prime is not as relevant. The mode of chromatography can affect the interpretation. For example: This is because size exclusion chromatography relies on the sample's interaction with well defined pores inside the support (inclusion/exclusion) to separate based on molar size. A variety of pore sizes can be used to "filter" the sample. So a large K prime value might be normal for a molecule that is low in molecular weight and spends a lot of time working its way through the column. A high molecular weight sample might just "shoot" through the column due to little or no interaction with the pores. You still need to have retention on the column, but now it is determined by how long it takes the sample to find its way out of the column. SEC columns are bracketed by Pore Size (e.g Mw. Excluding all samples that do not "fit"). With size exclusion columns, determine the Retention and Exclusion times, not the K prime). This article is specific to thr more common cases where traditional HPLC NP or RP modes are used. In these cases, low K prime values indicate no retention took place and the method fails all claims of specificity for the sample (selectivity is absent or poor). HPLC methods with little to no selectivity fail scientifically as no chromatography has taken place.

HPLC Peak Tailing - Some of the Most Common Reasons For it

Three easy ways to minimize chromatography peak tailing:

(1) Tailing often results from using “Type – A” HPLC silica. Type-A silica often contains more acidic silanol groups and metal impurities than Type-B. To improve peak shape, use modern “Type – B” silicas which are of higher overall purity, have less metal contamination and feature minimal silanol ionization under higher pH conditions.

(2) Minimize ionic interactions and utilize a buffer or ion pairing agent (e.g. TFA 0.02%) in your mobile phase. Select a buffer that is at least 2.0 pH units away from your sample's pKa and use the smallest concentration or amount that gets the job done. For LC/MS or MS/MS applications, remember to only use volatile buffers and avoid the use of ion pairing agents unless absolutely necessary (and if used, use at the lowest possible concentration to avoid source contamination).

(3) Always use a freshly washed and equilibrated column. Is the column fouled or the inlet frit dirty? If the head of the column is fouled from sample overloading or from a failure to wash off strongly retained compounds from many runs (much more common problem), then your peak shape and reproducibility will suffer. Incorporate a washing step in between your analysis methods which utilizes a solvent which is stronger (in concentration) than your mobile phase to wash off any strongly retained material after each run. For example, if you normally end a method with an 80% concentration of ACN, utilize a separate wash method which has 95% ACN in it. Allow enough wash time for this work.

Standard Addition HPLC Calibration Method (Spiking)

The standard additions calibration method is used to determine the concentration of an analyte which is in a sample matrix such as is commonly found when working with clinical, biological or food samples. To rule out interference from other components in the sample matrix, the sample is “spiked” and the detector response is monitored for any change which is not due to the change in sample concentration. The fortified standards created by spiking the original samples is useful when no version of the sample matrix is available without the analyte for use as a true blank. 

Common “Spiking” Standard Addition Procedure Example:

Divide up your sample evenly into FIVE volumetric flasks, representing five different final concentrations of spiked samples. Into four of the flasks add the same volume of increasing linear levels of the analyte to create four different calibration levels (e.g.  spiked amounts such as 25, 50, 75 and 100 units). Into the fifth flask add the same volume of diluent, without any spiked component, to serve as the ‘0’ or blank equivalent std. Next, extract all samples, inject and analyze the data per the usual method. A calibration curve of this new data set should show a linear relationship. If so, and your method is validated, you may be able to use just one spiked sample along with your regular samples to show that the reported concentration of your sample is not effected by the matrix and save yourself a lot of work (because you would need to prepare these standards and run a calibration curve each time).