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The “Breakdown” of Endrin and DDT for Non-Environmental Chemists

As the title makes clear, this post is aimed more at non-environmental laboratory personnel who are not familiar with US EPA methods.  If you are one of those environmental lab gurus who shudders at the sound of a “pesticide degradation check”, this is probably stuff you already know.

Anyways, for the rest of you, you may have noticed that a lot of chromatography consumable manufacturers, like Restek, like to give you performance specs for endrin and DDT breakdown in marketing literature, application notes, etc.  Some of you may wonder what this means or why it matters to you.

Endrin

Endrin

To start off, endrin and DDT are two chlorinated pesticides, whose usage has been banned or regulated in many countries, along with several other chlorinated pesticides.  These pesticides largely came into use after WWII and became widely used due to their effectiveness as insecticides.  Most of you are probably aware of DDT and its historical use in fighting malaria carrying mosquitos.  Several of these pesticides have persisted in the environment for long periods of time without degrading.  They also are not easily broken down by biological systems, causing a buildup in fat cells that can be passed through the food chain.  Rachel Carson released her famous environmental science treatise “Silent Spring” in 1962, which specifically focused on the dangers of pesticide accumulation in the environment, with many references to DDT.  The book helped to spawn a large scale environmental movement and changed the way we think of chemicals and the environment forever.  Eventually, growing environmental concerns would lead to the creation of the US Environmental Protection Agency (EPA) and result in greater legislation and regulation over chemicals in the environment.

4,4'-DDT

4,4′-DDT

This brings us back to endrin and DDT, which would be phased out of use, along with other chlorinated pesticides.  Even though not in use today in the US, these chemicals still persist in the environment.  The US EPA has developed analytical methods to analyze for a multitude of environmental contaminants from chlorinated pesticides to disinfection by-products.  These analytical methods are designated by number codes, such as 8081B, 8270D, 525.2, 608, etc.  One specific EPA method for analyzing for chlorinated pesticides by gas chromatography is 8081 (8081B, being the most current revision).  The method outlines requirements for looking at samples by GC, using dual columns with electron capture detection.  EPA methods are often explicit in many quality control requirements, such as how many points the calibration curve must be, how often a calibration check must be run, requirements for blanks, matrix spikes, etc.

One of the specific requirements of EPA method 8081 is a pesticide degradation (breakdown) check prior to running samples and at least every 12 hours during a sequence of samples.  This check involves injecting a mid-level standard containing only endrin and DDT and calculating what is known as the “breakdown” percentage.  DDT degrades to form DDE (dehydrochlorination) and DDD (dechlorination) and endrin degrades to form endrin aldehyde and endrin ketone (epoxide group in endrin is opened to form either aldehyde or ketone).  These reactions can easily occur at high temperatures, like those found in the inlet, as the compounds interact with a variety of surfaces and matrix components.  Breakdown percentage is calculated as follows (area refers to peak area):

Breakdown_EquationEPA method 8081B requires each breakdown percentage to be below 15% to continue with the analysis of actual samples.

endrin_ddt_cgram_sky

This pesticide degradation check is used as an indicator of instrument cleanliness/inertness.  The goal of any chromatographic method should be to detect only what is found in the initial sample that is injected into the instrument (easier said than done!).  As the instrument gets dirty from sample matrix or if you do not use quality inert consumables (liners, columns, press-tights, etc), breakdown of these very sensitive pesticides will occur in the GC system.  This can compromise the analysis, as products are being formed within the instrument rather than actually coming from the sample.  The breakdown products for endrin and DDT are also compounds of interest in method 8081, since breakdown occurs naturally in the environment, as well.  Some of the breakdown products are more toxic than the parent products to biological systems.

These particular pesticides were chosen because they are extremely sensitive to chemical change within a hot injector and require very inert conditions to perform well.  The performance of these pesticides has implications beyond that for themselves and their breakdown products.  Poor performance (aka high breakdown) for these pesticides is a likely indication that there will be similar issues with other sensitive analytes as well.  Even if you don’t work in an environmental lab and perform this degradation test, the breakdown numbers can still be meaningful.  Inert liner deactivations like the Sky deactivation involve complex chemistry to combat analyte interaction and breakdown in the inlet.  While you may never personally analyze endrin or DDT, the low breakdown percentages that you see in product literature are indicative of a quality deactivation that will work well for a large number of sensitive analytes at trace levels.

Link to US EPA Method 8081B: 

http://www3.epa.gov/epawaste/hazard/testmethods/sw846/pdfs/8081b.pdf

A quick evaluation of some disadvantages to performing semivolatiles analysis by EPA Method 8270D with splitless injection

This blog is part of a series; the previous installment can be found here.

Figure 1 - The later eluting PAH peaks are displaying the characteristic exaggerated shark fin shape of significantly overloaded peaks when analyzing the 8270 MegaMix at 120 ng on column by splitless injection.

Figure 1 – The later eluting PAH peaks are displaying the characteristic exaggerated shark fin shape of significantly overloaded peaks when analyzing the 8270 MegaMix at 120 ng on column by splitless injection.

Semivolatile calibrations on the 30 m x 0.25 mm ID x 0.25 µm df column format often range from 1.0 to over 100 ng/µL; however, a 0.25 mm ID column usually experiences peak overload as the mass on column approaches 10 ng. Column overload presents initially as slight peak fronting, skewing the tailing factor of a normally Gaussian peak to < 1.0. As mass on column continues to increase throughout the calibration range, isobars that elute close together—such as benzo[b]fluoranthene and benzo[k]fluoranthene— quickly become unquantifiable. Given the geometric nature of the typical calibration scheme, calibrations acquired under splitless conditions can quickly transition beyond acceptable chromatographic performance. Figure 1 clearly highlights the exaggerated shark fin appearance of severely overloaded peaks resulting from the splitless analysis of 1 µL of 120 µg/mL 8270 calibration standard. Peak shapes were so poor across the board for the 120 µg/mL calibration standard under splitless conditions that I dropped the level from the calibration, which has a clear impact on calibration dynamic range, as shown in Table 1.

Table 1 – Evaluation of the less volatile range of an 8270 calibration. The solid blue boxes indicate a dropped calibration point while a ‘*’ indicates the point was used (for easy counting).

Table 1 – Evaluation of the less volatile range of an 8270 calibration. The solid blue boxes indicate a dropped calibration point while a ‘*’ indicates the point was used (for easy counting).

Furthermore, the resolution of closely eluting isomeric pairs is particularly at risk when the column is overloaded because fronting causes the separation to collapse. Section 11.6.1.4 of EPA Method 8270D states that structural isomers are sufficiently resolved and may be reported individually when the valley height (hv in Figure 2) is less than 50% of the average peak height. Insufficient resolution requires reporting the isomers as a pair with combined results. The most common critical separation that requires reporting as separate values for each compound is the benzo[b]fluoranthene and benzo[k]fluoranthene isomeric pair. Conversely, the coelution of 3-methylphenol and 4-methylphenol is a good example of insufficient resolution requiring the reporting of a combined result.

Figure 2 - EPA resolution criteria indicates that hv must be less than 50% of the average hp for the two peaks.

Figure 2 – EPA resolution criteria indicates that hv must be less than 50% of the average hp for the two peaks.

Take a look at how the benzo fluoranthene resolution held up across the 9-point splitless injection calibration range of 0.10 to 120 ng on column (Figure 3). The three highest concentration calibration standards (120, 80, and 40 µg/mL) do not meet the 50% valley resolution criteria. The peak fronting and resulting overlap from column overload make it impossible to generate a linear calibration including these points. Looking at Table 1, you can see that we dropped several high points for all the closely eluting PAH isomeric pairs (phenanthrene and anthracene, fluoranthene and pyrene, benz[a]anthracene and chrysene, and benzo[b]fluoranthene and benzo[k]fluoranthene) as well as indeno[123-cd]pyrene and dibenz[ah]anthracene (each has a minor product ion that interferes with the quant ion of the other).

Figure 3 – This overly of extracted quant ion chromatograms illustrates how benzo[b]fluoranthene and benzo[k]fluoranthene resolution rapidly collapses as the column overloads.

Figure 3 – This overly of extracted quant ion chromatograms illustrates how benzo[b]fluoranthene and benzo[k]fluoranthene resolution rapidly collapses as the column overloads.

Figure 3 also highlights the retention time drift that occurs as mass on column increases. The peak apex of benzo[b]fluoranthene shifts more than 0.2 minutes across the calibration standards. Section 11.3.5 of EPA Method 8270D defines the maximum acceptable retention time drift across a calibration as 0.06 relative retention time (RRT) units (normalized by the internal standard retention time). The RRT drift for both benzo fluoranthene isomers was 0.01, clearly within method specification, but hardly optimal. While the target peaks are easy to identify during evaluation of a continuing calibration verification (CCV), wandering retention times combined with multiple non-target peaks from coextracted material will make target peak identification significantly more difficult during real sample analysis.

Here’s to HILIC: tailor-made for the MS analysis of polar analytes and highly water-soluble species

There is much discussion these days about Hydrophilic Interaction Chromatography (HILIC) and the options it provides today’s analysts. And there should be.  It’s powerful for analysts working with polar metabolites or highly water-soluble compounds, namely when using MS/MS techniques. Since phase chemistry is Restek’s specialty, and we have designed our Raptor SPP LC column line for use with MS/MS, we’d like to illustrate how HILIC can help you.

If you look back to the beginning, the founder of chromatography, Mikhail Tsvet, set the stage for it all. In 1906, Mikhail established the first separation using what is now considered normal phase. It’s not the “norm”, but it was first. This was used for the separation of fat-soluble compounds using a polar stationary phase.  The elution order, as a result, was non-polar to polar.  Once scientists figured out how to attach a phase, like a C18, they then made something, well, opposite.  A non-polar stationary phase used in conjunction with a polar mobile phase yields the “reverse”, polar to non-polar elution; hence “reversed phase.” This marked the true beginning of liquid chromatography.   

Aqueous normal phase and HILIC fall between these two classic modes. They utilize a polar stationary phase and a less polar mobile phase. So, we get a mix of the two; the retention profile of normal phase with the use of aqueous mobile phases. For the mass spectrometrist, this is big. We can now retain polar compounds without getting too funky with the mobile phase. While all HILIC columns are either highly polar or more polar than a C18, the mobile phase does the work. More specifically, the mobile phase’s interaction with the silica does much of the work – without traditional additives and reagents that kill MS sensitivity. And bonus – stationary phase selectivity enhances HILIC-mode-friendly columns’ versatility and adds to the mechanisms available for complex retention needs.

How it works: mobile phase choice and gradient manipulation create the formation of an aqueous layer with the polar silica or phase support, and this allows the use of high organic solvent for a secondary partitioning mechanism to occur. It’s this magic that allows polar compound analysis using MS-friendly mobile phases.

Adding stationary phase into the equation widens the selectivity profile. The new Raptor FluoroPhenyl phase exhibits this versatility. While you can run in HILIC mode for compounds such as 4-Mei: http://www.restek.com/chromatogram/view/LC_FF0559, it also resolves difficult to retain Vitamin D metabolites or basic drugs like taxanes: http://www.restek.com/chromatogram/view/LC_BA0351

We view HILIC as a mode, rather than a technique. Modes are normal phase, reversed phase, ion exchange, HILIC, etc.; techniques are workflow- and instrumentation-based, and take into account more than the column and mobile phase. But neither of these is as important to consider when discussing HILIC as the retention mechanism. To consider retention mechanism, look at the analytes of interest for direction. Retention via cation exchange and polarizability mechanisms respond well to HILIC mode analysis. Nitrogen-containing analytes and Lewis bases are candidates. Detailed discussion on retention mechanisms can be found here: http://www.restek.com/pdfs/GNFL1318B-UNV.pdf

We believe the HILIC mode offers impressive versatility for the MS/MS analyst. It opens the door for polar analytes, water-soluble bases and species, and otherwise non-C18-amenable compounds – without extensive method development and additives. Be on the lookout for more mixed-mode and HILIC applications and products from Restek.

Learn more about Raptor™ SPP Core-Shell LC Columns

 

Split vs splitless injection GC-MS: A head-to-head evaluation of calibration performance on a Rxi-5ms GC column using EPA Method 8270 semivolatile organic standards and calibration criteria.

My colleagues and I have been extolling the virtues of split injection for a while now. We’ve demonstrated that fast sample transfer often yields better chromatographic results (especially for early eluting volatile compounds). We’ve established that starting oven temperatures can be elevated, shortening run time and increasing sample throughput. Most importantly, we’ve shown that inlet discrimination and adsorptive loss are minimized, even after running many dirty samples. Performing a head-to-head calibration evaluation on the same column while holding everything constant except inlet liner, oven program, and MSD gain factor clearly highlights the advantages of split analysis and the superior inertness of the Rxi-5ms.

Calibration Preparation and Evaluation

A 9-point calibration curve was prepared at 0.10, 0.50, 1.0, 5.0, 10, 20, 40, 80, and 120 μg/mL using Restek 8270 analytical reference materials (cat# 31886, 31888, 31063, 31850, 31852, 31879), establishing an on-column calibration range of 0.0091 to 11 ng for the split analysis calibration and 0.10 to 120 ng for the splitless analysis. The split and splitless calibration data were collected on the same instrument on sequential days using the same tune file, but with a gain factor of 3.0 with a precision split liner for split and a gain factor of 0.3 with a single taper with wool liner for splitless. The complete instrument parameters are listed in Table 1 for split analysis and Table 2 for splitless.

The average % RSD for the split calibration was 7.0, with only 2 compounds (2,4-dinitrophenol and benzoic acid) exceeding method criteria when evaluating by response factor (RF) % RSD. This was marginally better than the splitless calibration, which had an average % RSD of 9.5, and three compounds (2,4-dinitrophenol, 4,6-dinitro-2-methylphenol, and benzidine) exceeding method criteria for evaluation by response factor % RSD. Additionally, only 2,4-dinitrophenol failed to meet minimum response factor criteria (min RF ≥ 0.010) for the 1.0 µg/mL calibration level (0.091 ng on column); however, 2,4-dinitrophenol met method linearity requirements and minimum response factor requirements when evaluated from 5.0 to 120 μg/mL (0.45 to 11 ng on column).

A performance summary comparing split and splitless calibration performance data for a subset of compounds can be found in Table 3. The table was designed to make it easy to visualize dropped calibration points. If you are looking to maximize your dynamic range, split analysis has obvious benefits.

Table 4 highlights the injection-to-injection variability by evaluating the surrogate response factors. The surrogates are at the same concentration in each calibration point, minimizing the effects of activity on the results for the acids and bases. You’ll notice that aside from 2-fluorophenol, the % RSDs for the surrogates acquired under splitless conditions trend up as volatility goes down. This is likely due to sample transfer efficiency dropping as analyte volatility goes down. This is a symptom of inlet discrimination and is normally exacerbated as the inlet gets dirty as real sample extracts are run. This is exactly why split analysis is advantageous; using a split ratio of 10:1 with a column flow of 1.4 mL/min results in an inlet flow of approximately 15.8 mL/min. This is significantly faster than the inlet flow under splitless condition. The split analysis provides a very fast transfer of a narrow analyte band to the head of the column, minimizing inlet discrimination and loss of sample to the inlet surfaces due to activity or other mechanisms of adsorptive loss.

Table 1 - Agilent 7890A-5977C GC-MS Split Acquisition Parameters

Table 1 – Agilent 7890B-5977A GC-MS Split Acquisition Parameters

Table 2 - Agilent 7890B-5977A GC-MS Splitless Acquisition Parameters

Table 2 – Agilent 7890B-5977A GC-MS Splitless Acquisition Parameters

8270 calibration comparison

Table 3 – Head-to-head evaluation of calibration performance on a Rxi-5ms GC column using EPA Method 8270 semivolatile organic standards and calibration criteria. At a 10:1 split, mass on column is approximately 1/11th that of the splitless injection for the same calibration point. Solid blue blocks indicate dropped calibration points. This is to make it easier to visualize the dynamic range for each 8270 component

Table 4 - RSD comparison of the surrogate response factors for each of the 9 calibration points. Precision is good for both techniques, but the split analysis precision is clearly better.

Table 4 – RSD comparison of the surrogate response factors for each of the 9 calibration points. Precision is good for both techniques, but the split analysis precision is clearly better.

SDS’s for 22010, 22011, 22014 and 22015 (Indicating Oxygen & Moisture traps/filters)

Even though we are not required to include a SDS (MSDS) for most of our products (articles), we often get asked for them for our gas filters/traps (for disposal information). Included below are the SDS’s for the following Restek catalog numbers:

22010_ph_so_trp

22010 & 22011 (Indicating Oxygen traps/filters)

22010-22011 MSDS Restek

 

 

 

22015_ph_so_gca

22014 & 22015 (Indicating Moisture traps/filters)

22014-22015 MSDS Restek

 

 

Related links:

How do I dispose of used gas traps or filters?

Changes are coming to the MSDS; um, I mean the SDS

SDS (MSDS) for VICI® Mat/Sen® Gas-Specific Purifier Modules

Contents inside your baseplate trap

Indicating Oxygen and Moisture Traps-“Hey, it looks like my trap arrived partially spent”

Join next Pittcon a class on Method Translation in gas chromatography. Learn how easy it is to optimize and speed up the analysis while you keep the same chromatogram

2011-jaap-pasfoto4-smallIn Gas chromatography there is often a need to optimize separations using different column dimensions, different linear gas velocity, using a different detector or using a different carrier gas. If you want to get the same peak elution order (same chromatogram), you must make sure that the elution temperatures of components is kept the same. This is only possible using a different oven temperature program. To calculate this program, there are free calculation programs available on the web. In this course we will discuss the details of conversion of methods so you get the same chromatograms with the new method.

translation

Method translation can be used in many situations. Goal is to get the SAME peak elution order (chromatogram) as before.

In this half day course (course 62), we will discuss the basics of converting existing GC methods into a new (mostly faster) method, and aiming for the same separation / peak elution order. If column dimensions, linear velocity or pressure drop over a capillary column is changed, and without changing temperature program, the separation of many components will change. Some separations will be better, some will go worse. In order to keep the separation similar, one needs to adjust the oven temperature, to get the same elution temperatures. For this, one can use free calculation programs, available on the web. See: http://www.restek.com/ezgc-mtfc

The different options of these calculation programs will be explained and demonstrated. It is recommended to bring a laptop to join hands on experience.

Target audience:    This short course is intended for the user of GC equipment that is able and want to learn to modify methods to improve lab efficiency. For maximum result, some experience with GC separations and analytical chemistry is preferred

Find details of the course 62 here: https://ca.pittcon.org/Technical+Program/tpabstra16.nsf/SCoursesByCat/9D7559F3DDAC893A85257E6700325005?opendocument

This half-day course will be presented on Monday, march 07 from 13:00 – 17:00 hrs.

Hope to see you in Atlanta!

Pittcon Atlanta landing page: http://pittcon.org/

Ernst Bayer Award to Junior Scientist Marco Nestola

The Working Group “Separation Science” as part of the German Chemical Society (GDCh) tenders the Ernst Bayer award for an outstanding publication in the field of analytical separation techniques to young scientists.

The 2015 award winner, Marco Nestola, received the prize personally during the 26th PhD-Conference, realized by the WG Separation Science from 10th to 12th of January 2016 in Hohenroda, Germany.

ernstbayer (2)

Dr. Martin Vogel, Representative of the German Chemical Society, presents the Award to Marco Nestola

The Award Committee honored the publication “Universal Route to Polycyclic Aromatic Hydrocarbon Analysis in Foodstuff: Two-Dimensional Heart-Cut Liquid Chromatography–Gas Chromatography–Mass Spectrometry”, published in Anal. Chem., 2015, 87 (12), pp 6195–6203.

Abstract:

Abstract Image

Analysis of polycyclic aromatic hydrocarbons (PAHs) in complex foodstuff is associated with complicated and work-intensive sample preparation. Chromatographic interference has to be faced in many situations. The scope of the current work was the development of a highly efficient two-dimensional heart-cut LC-LC-GC-MS method. Detection was performed with a time-of-flight mass spectrometer (TOF-MS) to allow for a comprehensive evaluation of the obtained data in terms of cleanup efficiency. Additionally, routine detection was performed with single quadrupole MS. An easy and quick generic sample preparation protocol was realized as a first step. During method development, focus was given to optimizing HPLC  cleanup for complex foodstuff. Silica-, polymeric-, and carbon-based HPLC phases were tested. Coupling of silica gel to π-electron acceptor modified silica gel showed the best cleanup properties. A four rotary valve configuration allowed the usage of a single binary HPLC pump. Screening of several fatty and nonfatty food matrices showed the absence of unwanted matrix compounds in the cleaned-up PAH fraction down to the low picogram range using TOF-MS. Limits of quantitation (LOQ) were below 0.1 μg/kg for all EU priority PAHs. Recovery rates ranged from 82 to 111%. Validation data fully complied with EU Regulation 836/2011. Sample preparation was possible in 20 min. Interlacing of HPLC and GC allowed an average method runtime of 40 min per sample.

The work was realized at the University of Duisburg-Essen, where Marco is part of the Working Group of Professor Dr. Torsten Schmidt and in a collaboration with Axel Semrau GmbH

Restek Corporation is very pleased about the choice of the committees, for we have supported this work and other, comprehensive  LC-GC technique related works of Marco Nestola since years. The shown work could benefit from our Restek Rxi-PAH GC column as well as from our PAH Reference Materials, dedicated to European Food regulation demands, like the EU 15+1 PAH standard and the EU PAH Interference standard, which is used as a System Performance Test.

Extraordinary Young Scientist Marco Nestola has improved the hyphenation of LC and GC for various topics, wherein his MOSH/MOAH approach is also reflected by products in our portfolio, e.g. the MOSH/MOAH cutting standard. Please refer to “Accurately Determine Mineral Oil Hydrocarbons in Food Using Restek’s New MOSH/MOAH Reference Standard”

The practical and commercial realization of Marco Nestola’s work, e.g. the MOSH/MOAH LC-GC-System and the new PAH system, based on the work described above, the LC-GC-System PAK can be found at Axel Semrau GmbH, where you may also find Restek products as strategic core products inside.

26th PhD-Student Conference in Hohenroda

Every year since 1990 the Separation Science Working Group, part of the German Chemical Society (GDCh), chairs a platform for PhD Students, working on topics related to Separation  Science. This meeting gives a chance to a good part of these students to improve their capability in presenting their research results to a grand audience, using most modern presentation techniques. The audience is formed by other PhD-Students (this year the conference hosted 134 participants), their Professors and Industry Representatives.

The conference is organized by students of a related working group. This year the students from Prof. Oliver Schmitd’s working group at the University of Duisburg-Essen did an outstanding job in upfront organization, but also in realization. Special thanks to Amela Bronja and Simeon Horst who were the faces of that wonderful Organization Committee.

For the conference venue is located out somewhere in the field, the entire group usually stands together during three days and two evenings, with a lot of chances for networking and scientific or private conversation.

Sponsors and facilities

Sponsors and Facilities

The conference program was divided into several sections (Liquid Chromatography, Gas Chromatography, Mass Spectrometry, Bioanalytics, Capillary Electrophoresis) and saw 26 scientific presentations. It is always exciting to recognize the high quality of the work, provided by the Junior Scientists and their working groups.

The presented topics are a cross section through all topics to be developed with a separation science technique, from Food Fraud (green harvested Ananas vs. freshly harvested Ananas) to Non Target Screenings in surface water or an attempt of a complete description of different Crude Oils, but also showing the trends in hyphenating Chromatography to new Sample Prep techniques, sophisticated detection techniques and miniaturization (HPLC on a chip, Multidimensional Micro-LC) .

Since ten years, Restek is awarding the three best presentations, chosen by the audience. The Student Award is part of our commitment to support young scientists as part of our future. This year, also BGB, the Restek partner in Switzerland and Austria, stepped in with an additional award for the best presentation. All three award winners are on top honored by the Springer publishing house with a book voucher.

Award Winners and Award Sponsors

Award Winners and Award Sponsors

The this year award winners are:

  1. place: Johanna Hoffmann, Fritz-Haber-Institute of the Max Planck Society, Berlin (WG Pagel). Presentation Title: Identification of Carbohydrate anomers using Ion Mobility Spectrometry
  2. place: Carsten Lotter, University of Leipzig (WG Belder). Presentation Title: HPLC-MS in Glas Chips
  3. place: Tobias Bader, Zweckverband Landeswasserversorgung, Langenau (WG Schulz). Presentation Title: Strategies for increasing the reproducibility of Non-Target-Screenings via HPLC-HRMS

Restek Corporation congratulates all winners and all presenters for their outstanding work. The program of the conference can be found here

One additional presentation is always given by a former conference attendee who has now made his way into a job, for most of the conference participants are close to finish their work and are shortly before entering their first job.

So it is not surprising that companies are using this meeting as a recruiting area. Several Job Offerings were presented by Chemical Industry representatives and from considerable Instrument Manufacturers.

 

Gruppe1 (2)

The attending PhD-Students and guests

 

Calculating pH

I’m guessing that most of us that work in the lab would find it more convenient to measure a pH rather than to calculate a theoretical value.  But, at times, it can be very useful to make that calculation and get an idea of what to expect.  As far as chromatography is concerned, all columns have suggested operating ranges for pH, so it is important to know whether or not it is safe to inject your sample. Of course, this pertains more to reverse phase LC, because sample extracts usually do contain at least some water.  (In fact, pH can only be measured in the presence of water to allow for dissociation.)

Here is what you need to know to calculate pH:

  • For weak acids or weak bases, the pKa of the acid or compound in solution
  • The molarity (M) of the solution, which is defined as moles per liter
  • If concentration is only known in weight units/volume, you will need to know the molecular weight of the acid or compound.
  • If concentration is only known in terms of volume/ volume, you will need to know the density of the acid or compound.

For strong acids

There could be a need to calculate pH as well for strong bases, but acids are much more commonly used with reversed phase LC, so our discussion here will be limited to the acids. Before calculating the pH, first determine the molarity of your solution.

Example calculation:  0.2% TFA (in water), v/v or volume/volume

Density =1.49 g/mL, Molecular Weight =114 g/mole

Molarity =2mL/1 L x 1.49g/mL x 1 mole/114g= 0.026 moles/L =0.026M

 

Then, determine the molarity concentration of H+ ions from the dissociated compound in solution. This is needed because pH is defined as the negative log of the concentration of H+ ions in molarity, expressed as [H+].

In the example above, TFA is a strong acid, so is 100% dissociated. In this case, the calculation is easy because the molarity for H+ ions is the same as the molarity of the acid.  It is 0.026M.

So, the pH is calculated for the example like this:

pH=-log (0.026)= 1.6

A good reference that can walk you through the calculations for pH and related topics can be found here at Purdue University’s website:

https://www.chem.purdue.edu/gchelp/howtosolveit/Equilibrium/Calculating_pHandpOH.htm

 

For weak acids or bases 

The example above is for a strong acid. To do this calculation for a weak acid or base, you would need to know the Ka, which is the acid dissociation constant for the acid, or the pka, which is the negative log of Ka. For this discussion, we will focus primary on weak acids, using the following equations:

Ka = [H+] [A-]/[HA]

pKa = -log Ka

Let’s do an example here for a monoprotic weak acid, in this case, a 1M solution of acetic acid.  If you look up the pKa for acetic acid, you will find that it is 4.754. So, using the above equations, we calculate:

pH for weak acid

 

 

 

 

 

 

 

 

 

 

 

 

 

So, now we know that a 1 M acetic acid solution has a pH of 2.38.

 

The next question you have may pertain to actual limits that we recommend for our columns. The following table lists some general guidelines for HPLC:

LC pH ranges

 

If you compare the above ranges to the example we did earlier for TFA, you can see that a 0.2% TFA solution is a bit too acidic (pH= 1.6) for most of our HPLC columns. It is easy to see why we do not recommend TFA as a mobile phase modifier very often. Also, I always try to remind folks to check their mobile phase periodically, as mistakes in preparation could lead to column damage if it should go unnoticed.

On the other hand, our result for 1 M acetic acid solution is too close for comfort to the lower pH limit for all of our fully porous particle columns. However, it should be OK for any of our Raptor columns.  Again, it is a good practice to check the pH and be certain no mistakes were made. 1M is actually higher than you will usually see for HPLC mobile phases. Usually for this reason, acetic acid does not present too much cause for concern.

 

For guidance with GC columns please see the following FAQ and blog post:

Can I inject acids or bases onto my polysiloxane column? Will they damage the column?

 

Capillary GC Column Killers- Part 1

 

I hope that you have found the suggestions and information here useful. Thank you for reading.

NJDEP-SRP Low Level TO-15 Series: Part 5 – Method detection limits…

If you recall from part 1 of this blog series, NJ LL TO-15 listed MDLs as being one of the items they modified from U.S. EPA Method TO-15. HOWEVER, the only “modification” I see made is that NJ LL has outlined “specific criteria that the laboratory must meet regarding the MDL and an MDL study must be conducted annually.” Other than that, NJ LL TO-15 actually states that “the laboratory shall calculate all Method Detection Limits (MDLs) in accordance Method TO-15. Section 11.2 of US EPA Method TO-15 requires the use of the procedures stated in Appendix B of 40 CFR 136 for performing the MDL study.”

So what does this mean? It means that we are basically doing what we always do for U.S. EPA Method TO-15, with the stipulation that we must conduct our MDL determination using a spiking solution at 0.20 ppbv and the derived MDL must be less than the Clean Canister Certification Level of… you guessed it… 0.20 ppbv. NJ LL does allow for some compounds to be spiked at higher concentrations, but we are going to skip over this cop-out, because we can get it done with the 0.2 ppbv. So using the parameters outlined in blog parts 2 and 3 of this series, we fire off 7 injections of our 0.2 ppbv standard onto the Markes CIA Advantage™ and here is what we get:

NJ LL TO-15 MDLs Final

Everything passes with flying colors! Yes, acetone and TBA were above 0.20 ppbv (marginally), but NJ LL already accounted for this and gave us some leeway by setting higher MDLs for acetone (1.00 ppbv) and TBA (3.00 ppbv). Remember that this is all done with full scan and only a 250 mL injection. Oh, and as usual these results were obtained on the same dirty source and without manual integrations (i.e., these are realistic results you should be able to easily achieve). Stay tuned for the final blog on NJ LL TO-15…