USP 281 and 731 in Under Half an Hour:

Determining Residue on Ignition (ROI) in Pharmaceutical and Nutraceutical Products by Gravimetric Loss on Ignition Analysis

Christopher Altamirano, Frank Kramer

Pharmaceuticals

Introduction

Pharmaceutical and nutraceutical drugs often consist of two main components: active pharmaceutical ingredients (API’s) and excipients.   API’s are the key active ingredients within a medicinal powder, capsule or tablet that aid in treating a disease or preventing illness.   Excipients are often natural or synthetic fillers used to add mass/volume, flavor and color to the delivery system of the drug.  Some excipients may even play important roles in prolonging the shelf life of the drug as well aiding in absorption into the body.   API’s are typically very low in concentration when compared to their excipient counterpart, making it difficult to uniformly produce drugs at consistent levels (see Figure 1).  The Food and Drug Administration (FDA) as well as the United States Pharmacopeia (USP) agencies have provided thousands of protocols and guidelines for pharma/nutraceutical companies to abide by to ensure uniformity in drug dosage.  Residue on Ignition or ROI is one such USP method (USP 281) designed to measure proportions of inorganic components of either the excipients, API or both within pharm/nutraceutical drugs.  These inorganic components include mineral salts and trace metals left behind after the product has been incinerated in a muffle furnace at 600 °C for several hours.

Figure 1

500 mcg API

Grains of salt simulates 500 mcg of API compared to tablet mass

 The traditional USP 281 method outlines a stringent and elaborate procedure to measure ROI which takes many hours of preparation, run time and cool down to complete.  Utilizing a gravimetric Loss on Ignition instrument capable of achieving 600 °C (added to the USP General Notices in 2011) allows the operator to replicate the temperatures of a muffle furnace without any preparation or cool down time.  Instruments such as the Computrac® MAX® 5000XL pair a sensitive four decimal place digital balance with the temperature performance of a muffle furnace (max temperature 600 °C).  This allows the operator to not only test for % ROI (i.e. % Ash) but also has the added benefit of analyzing percent moisture in a product at lower temperatures.  Additional functions such as linked test capability, slow ramp temperature scans and data acquisition from web server can be useful analytical tools when optimizing or validating the chemical composition of your final product.

Computrac MAX 5000XL

Computrac® MAX® 5000XL Moisture & Ash Analyzer

Utilizing both a  standard oven and muffle furnace protocols as references, the percent moisture and ROI were calculated from several vitamins and supplements and compared to samples analyzed by the MAX® 5000XL.  All products were either crushed by mortar and pestle or poured from their gelatin capsule.  All drugs analyzed were private-labeled grocery products which included Vitamin B12, C, D, and Red Yeast Rice Powder.

Results

Moisture Results

Moisture content is important for many products, but imperative for food and drugs.  If the moisture content is too high for a particular product, the risk of microbial contamination is greater, while if too dry, the product may become stale or ineffective.  Monitoring moisture content (as outlined in USP 731 Loss on Drying method) during drug formulation and after for quality assurance is useful in identifying problematic batches which saves time, money and the wellbeing of consumers taking the drug.  All tests (both references and MAX® 5000XL) were performed in triplicate.  Oven references were run with 3 grams of powdered sample at 100 °C for 1 hour.  The MAX® 5000XL was run under similar conditions.  Error bars are plotted to represent one standard deviation above and below the mean of each data set.

Figure 2a

Moisture Chart

The data from Figure 2a demonstrates the equivalence between the MAX® 5000XL and the standard oven method (USP 731).  For all supplements analyzed, each data set falls within the margin of error of the corresponding oven mean.  Moreover,  Figure 2b shows the averaged test time for each group of samples.  Test times are considerably shorter on the MAX® 5000XL when compared to the standard oven reference. 

Figure 2b

Moisture Test Times

Residue on Ignition Results

If both moisture and ROI analysis were to be performed on the same sample, it would involve running a 1-hour test in the oven @ 100 °C, plus linking the same set of samples to a 2-hour furnace method at 600°C.  When considering crucible purge time, run time, cool down town and time to record the difference in weights for both sets, the total test time would be between 4-5 hours.  Fortunately, the MAX® 5000XL has the capability of linking multiple tests (e.g. Moisture and ROI) on the same sample.  Therefore, more information can be gathered in a shorter amount of time without excessive operator involvement.  Moreover, the instrument allows the operator to calculate Total % ROI or Dry % ROI within a series of linked tests. All tests were performed in triplicate.  Muffle furnace references were run with 3 grams of powdered sample at 600 °C for 2 hours.  MAX® 5000XL was run under similar conditions. As in the graph for the moisture test, error bars are plotted to represent one standard deviation above and below the mean of each data set.

Figure 3a

ROI Chart

The data from Figure 3a demonstrates the equivalence between the MAX® 5000XL and the muffle furnace method (USP 281).  For all vitamin/supplements analyzed, each data set is ± the standard deviation of the corresponding muffle furnace.  See figure 3b for test times.

Figure 3b

ROI Test Times

Analytical Temperature Scans

Infrared Thermal Image of MAX 5000XL

Infrared Thermal Image of MAX® 5000XL

Along with moisture and ROI analysis, the MAX® 5000XL allows the operator to control the temperature rate with a resolution of 1 °C/min.  These temperature ‘scans’ may take longer than the typical ROI tests but allow for separate volatiles to evolve from the sample at different temperatures.  This form of analysis is considered a thermo-gravimetric analysis.  When all four drugs were scanned separately with a rate of 5 °C/min from 50 °C to 600 °C, the resulting graphs were produced (Figures 4a & 4b).

Figure 4a

ROI Scan

Figure 4a demonstrates that the % ROI scan for all four vitamins/supplement have different thermal profiles.  It is important to note that with the exception of the Red Yeast capsule, most of what is being incinerated is not the actual API listed (since it is in such low concentrations) but rather a mix of different excipients.  Excipients are provided for each vitamin/supplement in Figure 5.  Knowing the thermal profile of each product may be useful in validating the correct proportions of each of these excipients. 

 Figure 4b

ROI Rate Scan

Figure 4b presents the rate profile during this temperature scan.  It becomes evident that each peak may correspond to a certain chemical species reacting to specific temperature.  Some peaks are even shared between different vitamins, which is not surprising since these vitamins/supplements were created by the same company using some of the same excipients (Figure 5).

Red yeast was chosen among the rest of the vitamins as a pseudo control supplement to compare against since there are very few excipients within the Red Yeast Rice Powder. The thermal profile reflects this purity since only two main peaks are observed: ‘moisture’ peak at 80 °C and the ‘API’ (red yeast rice powder) peak at 300 °C.  Although these rate profiles offer insight into the chemical proportions of these vitamins, they do not necessarily describe a specific chemical species.  Further investigation would need to be performed to deduce this information from the thermal profile.

Figure 5

Excipient List

Conclusion

Pharmaceutical and Nutraceutical companies alike have a responsibility to ensure the drugs they are distributing are not only safe and effective for the consumers but are consistent between different batches and manufactures.  In order to ensure this uniformity in their product, many FDA and USP regulations should be considered during and after the formulation of the final product which includes measuring the residue on ignition (ROI) of the excipients and API’s.  The % ROI describes the amount of total concentration of inorganic salts and trace metals that fail to ignite at 600 °C.  Arizona Instrument was able to demonstrate equivalency between the traditional muffle furnace method and the Computrac® MAX® 5000XL Moisture and Ash Analyzer for several vitamins and supplements.  Along with % ROI, % Moisture and thermo-gravimetric analysis was also performed with minimal sample handling for the operator and in a timely fashion.  The MAX® 5000XL has proved to be useful in data acquisition and graphical representation, which can be downloaded through the Web Server.  This data can be graphed to produce true analytical representation of each thermal profile for each vitamin/supplement.

In this investigation, only 4 vitamins/supplements were analyzed, but many other foods, drugs, and excipients have been analyzed by the same method.  Arizona Instrument and the Computrac® MAX® 5000XL have included elements of a 4 decimal place digital balance, standard oven, muffle furnace, and a TGA instrument to create a durable instrument that yields accurate and reproducible results.  Whether it is in a Research and Development laboratory, Quality Assurance laboratory or used on the Production floor, the MAX® 5000XL will be a useful analytical tool for any pharmaceutical or nutraceutical product.

What is a Bottle Purge and Why do You Need it?

Vapor Pro

The Computrac® Vapor Pro® series (Standard & Rx) have proven their worth as reliable and accurate moisture-specific instruments used in a variety of different industries.  The Vapor Pro® interface is very easy to use and program with only a handful of testing parameters required to accurately analyze your sample for moisture content.  Testing parameters include test temperature, ending criteria, start weight input method, and a bottle purge option (in seconds); but what does a bottle purge do?  If you have asked yourself this question, you are not alone.  This adjustable option may not be intuitive, but imagine an empty sample vial sitting on your bench top.  If you were to cap that empty vial and run it as a mock sample with a ‘0 sec’ bottle purge you might expect 0 µg of water to come from an empty vial right?   The answer might surprise you since the vial is not actually empty!

Although the vial may look dry and void of condensation, there are actually microscopic droplets of water adhered to the sides of the vial.  Moreover, as you cap the empty vial, you are actually trapping the relative humidity from the environment within the vial.  If this humidity is not accounted for, you could be inadvertently adding moisture to your sample during testing.  So how much moisture is in an empty vial?  This may depend on your local and seasonal humidity, however normal levels range from 200-400 µg of water! The function of the bottle purge is to rid the vial of this moisture prior to analyzing your sample’s true moisture.  The empirical data below presents several empty vials run with varying bottle purges with their corresponding moisture levels.  Arizona Instrument typically recommends a bottle purge from 30-45 seconds (although exceptions may be made depending on sample type).  If you are unsure of what bottle purge to use for your sample, feel free to contact your Account Representative at AZI for more information.  Your Account Representative can arrange for samples to be sent to the Sales Test Lab for testing to give you an optimized set of parameters including a recommend bottle purge time.

Bottle Purge  Figure

AZI

Mercury Vapor Analyzers: Finding the Right Fit for Your Needs

Christopher Altamirano, Lab Manager

Broken Thermometer

The toxic effects of mercury vapor on human health have been well documented throughout the years.  Agencies such as the Environmental Protection Agency (EPA) regulate and set strict limits on the amount of mercury vapor which can be present in air: 1µg/m3 for residential properties, and 25µg/m3 for industrial properties.  To ensure compliance with these regulations, industrial hygienists, clean-up crews and government agencies must rely on portable instrumentation to measure mercury vapor levels on site.  There are many different technologies which can be used to detect mercury, and some may be more appropriate in certain environments than others.  This paper will explain differences between gold film sensors, atomic absorption spectroscopy and atomic fluorescence spectroscopy, focusing on how these technologies work, what interferences they have and how sensitive they are to low levels of mercury vapor.

Gold Film Sensor Technology

How it works

Gold Film Sensors were the first reliable forms of mercury detectors due to gold’s affinity for elemental mercury.  Arizona Instrument (AZI) took advantage of this affinity and coupled it with gold’s inherent electrical conductivity to create the Jerome J431 & J405 mercury vapor analyzers.  If a mercury rich air sample is swept over a thin gold film, the mercury will deposit on the gold and change the electrical resistance of the foil.  This change in resistance is directly proportional to the mass of mercury vapor taken from a known volume of air, which can be calculated in mg/m3.  If the gold becomes saturated over time, the instrument offers a ‘regeneration’ feature that bakes the foil at an elevated temperature where the mercury deposits are vaporized and collected in the scrubber.  The schematic below demonstrates how this works.

Gold Film Sensor

Interferences

The ‘green’ box above represents the ‘Acid Gas Filter’, an activated carbon filter designed to remove hydrogen sulfide gas.  Hydrogen sulfide (and mercaptans) along with ammonia and chlorine gas will react with the gold film and produce a false positive.  However, along with the internal gas filter, several other external filters can be purchased from AZI to remove ammonia and chlorine gas if it ever poses a major interference without reducing the mercury concentration in your sample.

J431

Jerome® J431 Gold Film Mercury Vapor Analyzer

Sensitivity

Arizona Instrument has been manufacturing its patented Gold Film Sensor mercury vapor analyzers for over 30 years, during which time the technology has proven to be effective for diverse applications and detection limits.  The Jerome® J405 has many updates from the older J431 model, but both are useful for different detection limits.   The J431 has a detection range from 3µg/m3 to 999µg/m3 with a resolution of 1µg/m3.  This detection limit falls just short of the EPA residential specification but is well below the industrial specification of 25µg/m3.  This instrument is better suited for industries concerned with exposing their employees and surrounding residents to harmful mercury vapor.

J405

Jerome® J405 Gold Film Mercury Vapor Analyzer

The Jerome® J405 is our latest gold film analyzer, equipped with an on-board data logging (20,000 data point storage) system and an optional USB data communication port.  The J405 has a detection range from 0.5µg/m3 to 999µg/m3 with a resolution of 0.01µg/m3.  This newest model of gold film MVA allows you to adhere to both industrial and commercial mercury regulations because it can read below 1µg/m3.  Both units offer continuous modes for surveying potential hot spots in the field, and are robust enough to withstand daily use in challenging environments.

Environments of Likely Interferences

Gold film interferences include hydrogen sulfide, ammonia and chlorine, however internal and external filters are available to remove these chemicals from interfering with your analysis.  One potential environment where gold film would not be ideal is an environment rich in both ammonia and chlorine.  Since only one external filter can be used during analysis it would be difficult to measure mercury without getting a signal from either the ammonia or chlorine.  Another potential environment not suitable for gold film would be an environment completely void of oxygen.  The gold film sensor requires the presence of some oxygen to be effective.  Since most industrial hygienists are measuring in environments where personnel could be exposed, it is unlikely that any environment would be void of oxygen.

Atomic Absorption Spectroscopy

How it works

Cold Vapor Atomic Absorption Spectroscopy (CVAAS) is another method used for mercury detection.  In mercury CVAAS, a light source of known wavelength and intensity (~254nm, middle ultraviolet spectrum) is radiated through a sample of air where the light eventually encounters a detector.  If mercury is present, electrons from within the mercury atoms will absorb some of this energy from the light source.  The difference between the initial energy of the light source and the energy measured by the detector gives you an indirect measurement of how many mercury atoms were present.  The schematic below demonstrates the path of the radiated light.  Several mirrors and photo multiplier tubes (PMTs) are used to amplify the signal difference.

Atomic Absorbance

Indirect method relies on how much energy was absorbed

Interferences

Unfortunately, atomic mercury in CVAAS is not the only chemical species to absorb this wavelength.  Many other substances can also absorb this wavelength and produce false positive readings.  The chart below lists a handful of some of the known interferences for CVAAS.  It is important to note that ‘hydrocarbons’ is a very broad description of many different forms of organic compounds.

CVAAS Interferences

Along with these interferences, the reflective mirrors could also become dislodged, soiled by material condensation (including humidity) or degraded by surface corrosion. 

Sensitivity

Because atomic absorption spectroscopy measures on the atomic level, the sensitivity range is lower.  Portable mercury CVAAS analyzers such as the Nippon® EMP-2 and the Lumex® 915 M claim to offer a low-end sensitivity of 0.1µg/m3 and 0.002µg/m3 (respectively), both of which fall below the EPA regulations for residential specifications.  However, with positive interferences being such a problem for CVAAS it is more than likely the instruments will be analyzing chemical species other than the intended mercury vapor (especially at that low level).  If the readings are taking place in a clean and dry environment, more merit could be given to the results.  However, most environments where mercury vapor analysis is required (laboratories, landfills, industrial chemical facilities) are rarely void of the interferences listed.  Moreover, the delicate placement of the internal mirror system may not be robust enough to be brought into a hazmat situation.

Environments of Likely Interferences

When using atomic absorption spectroscopy to detect low levels of mercury contamination, it is imperative that  your instrument is detecting a true signal and not just ‘noise’ from other chemical species.  An example of a common low-level mercury analysis application is the process of decommissioning laboratories and hospitals.  These old buildings have been abandoned for many months or years and have collected a fair amount of dust and construction debris.   Before demolition can begin, the entire square footage of the building must be below a certain limit of mercury vapor.  It is difficult to confidently measure low-end mercury vapor over dust and smoke if the environment you are sampling from is substantially contaminated with these particulates in the air.

Mercury analysis in the petroleum processing industry is also quite common.  Industrial hygienists must monitor the naturally occurring mercury levels emanating from crude oils wells and processing plants.  Common interferences with low-level mercury analysis using atomic absorption in this industry are petroleum hydrocarbons.  Crude oil aromatic hydrocarbon vapors along with the toluene/xylene co-solvents used in processing are major sources of interferences for atomic absorption spectroscopy.  Many double bonds and resonance electron structures within these hydrocarbons absorb ultraviolet light and would be measured as a mercury vapor.  Because of these interferences, atomic absorption spectroscopy is not a suitable fit for mercury vapor analysis in this industry.

Atomic Fluorescence Spectroscopy

How it works

Atomic fluorescence and absorption are two terms that are related but have two different meanings.  Cold Vapor Atomic Fluorescence Spectroscopy (CVAFS) is an improvement upon the traditional CVAAS.  When a mercury atom absorbs the energy from the UV wavelength, an electron transitions from a stable ground state to an unstable ‘excited’ state.  This excitation event describes the atomic absorption as discussed in the previous section.  However, when the energy source is removed the excited electron returns to its ground state.  In doing so, a photon of light is emitted during the loss of potential energy.  This fluorescence of light is often unique for various chemical species.  Mercury in particular absorbs light at 254nm and fluoresces light at the same wavelength.  Because the light absorbed and emitted are at the same wavelength, this form of fluorescence is referred to as resonance fluorescence. Other chemicals such as chlorides, sulfides and hydrocarbons absorb light at 254nm but either do not fluoresce or fluoresce at a different wavelength.

J505

Jerome® J505 Atomic Fluorescence Spectroscopy Mercury Vapor Analyzer

Arizona Instrument took advantage of the unique resonance fluorescence of mercury to detect ultra-low concentrations of mercury vapor while minimizing the interferences involved with atomic absorption alone. The Jerome® J505 Atomic Fluorescence Spectroscopy Mercury Vapor Analyzer is the first hand-held instrument of its kind.  Although other chemical species may still absorb the energy from the light source, the J505 only detects the specific wavelength that is fluoresced radially from an air sample.  The amount absorbed is inconsequential because the mercury concentration is revealed by the amount of light fluoresced at a 90° degree angle.  This technology is a more direct method of analysis since the instrument is quantifying individual photons of excited mercury atoms in a sample.  The diagram below outlines how this done without having to amplify the signal through a series of mirrors.

Atomic Fluroescence

Interferences

Because the J505 only measures radial resonance fluorescence of 254nm, only a chemical species that is excited at 254nm and then fluoresces at 254nm will be measured.  This stringent criteria eliminates nearly all sources of interferences ensuring you get accurate and repeatable results in the field.  The only positive interference ever to be reported is a high concentration of acetone vapor.  Fortunately, most areas of potential mercury contamination are void of such levels of acetone vapor.

Sensitivity

The J505 has a detection range from 0.05µg/m3 to 500µg with a resolution of 0.01µg/m3, which is well below the EPA, OSHA, and NIOSH standards for ultra-low mercury vapor specifications.  Moreover, the J505 does not share any common interferences with traditional CVAAS technologies.  The J505 is a robust analytical tool that can be used in a variety of fields.  There are no amplifying mirrors like the CVAAS units and unlike the gold film sensor instruments, no regeneration is ever required.  The instrument stores up to 10,000 tests on its internal memory board, which can easily be downloaded from a USB port.  Like the J405, one time or continuous sampling can be performed on the instrument to aid in searching for hot spots of contamination.

Environments of Likely Interferences

Atomic fluorescence spectroscopy selects for a very narrow criteria of chemical species that can absorb and fluoresce at the 254nm wavelength.  Industries which use large quantities of acetone as a solvent could potentially present environments in which the J505 might have difficulty exclusively detecting mercury vapor.  Such environments could potentially be encountered in certain types of chemical processing plants or paints and coatings settings. 

Conclusion

Arizona Instrument takes pride in the instruments we manufacture and wants to educate our current and future customers on what form of analysis might be appropriate for their needs.  In this paper we covered three very different technologies of mercury vapor analysis.  Gold film is a proven, reliable form of low and high-end mercury detection with few interferences (hydrogen sulfide, chlorine, and ammonia).  However, each of these interferences can be filtered away without changing the accuracy of your results.  The indirect detection of atomic absorption spectroscopy was shown to be useful at ultra-low levels of mercury analysis, but with many different types of interferences that cannot be filtered.  This form of analysis raises the question: if there are so many potential interferences, how can you be sure your signal is really mercury?  The solution to this problem was designing an instrument with very strict parameters for mercury analysis.  Utilizing the unique resonance fluorescence of mercury, the Jerome® J505 is able to directly measure mercury atoms during their excitation phase.  Not one method is perfect, but an understanding of how the available technologies behave differently in your unique application setting will be a key factor in making the decision that is right for you.

Appendix: Mercury Vapor and Human Health

Elemental mercury (Hg) is the only metal on the periodic table that remains in its liquid phase under standard temperature and pressure (STP); giving it the appropriate nickname of ‘quicksilver’.  Many of us may envision this form of mercury as this shiny silver puddle evading capture, but mercury can be found in many other forms in our everyday lives.  Fluorescent lighting, antique switches, dental fillings and thermometers are just a handful of items that contain the toxic metal, not to mention its many uses in industrial processing (chlorine, cement, and gold purification).  Most of these items are sufficient at sequestering or minimizing the exposure of the dangerous metal, but accidents can and do happen.  Thermometers break, old bulbs and switches are crushed in landfills, and industrial mercury incidences can occur.  So why is it so bad for us to be exposed to this metal?

Mercury Puddle

Elemental mercury has an unusually high vapor pressure for a metal (0.0018 mm Hg) at room temperature which corresponds to approximately 2.4 part per million.  Studies have shown that skin contact and ingestion are dangerous methods of exposure, but inhalation of mercury vapor is perhaps the most lethal form of absorption.  Symptoms of mercury exposure include seizures, dementia, and in some cases even death.   Because of these risks, several guidelines and regulations have been developed to limit the amount of mercury people can be exposed to along with special methods for cleaning up mercury if an accident should occur.  Currently, the time weighted average limit for mercury varies depending on regulating agency.  For OSHA, the limit is 0.1mg/m3; NIOSH sets the limit at 0.05mg/m3; while ATSDR sets its limit at 0.001mg/m3.  Because these agencies all differ in application and exposure limit, the environmental protection agency (EPA) has a standard minimum limit of mercury exposure to 1µg/m3 for residential and 25µg/m3 for industrial properties.

 

Purity and Poison Gas: Investigating the Relationship between Gypsum Purity and Hydrogen Sulfide Production under Anaerobic Conditions

Christopher Altamirano, Lab Manager

Introduction

Calcium Sulfate (CaSO4) more commonly known as gypsum is a naturally occurring white crystalline mineral that has many different uses and applications.[1]  When water is added to gypsum, the resulting gypsum paste can be molded and shaped into various forms and dried to maintain its shape.  The word ‘gypsum’ is derived from the Greek word for plaster, which is telling of its initial use as wall plaster during the ancient Greek and Roman empires.[2]  In the late 1800’s gypsum paste was molded between two sheets of paper board and dried to create the first functional wallboard.[3]  Gypsum in its hydrated form (CaSO4·2H2O) is used in the construction industry for fire resistance in buildings.  Outside of the construction industry, gypsum is used in the food and fertilizer industry for calcium and sulfate fortification respectively.

Although gypsum has many applications in various fields, when it is eventually deemed unusable, it ends up in a landfill along with other construction debris.  These construction and demolition (C&D) landfills promote a low oxygen environment (anaerobic) for any product buried deep within the fill.[4]  When gypsum is buried within the pile it is metabolized by specialized sulfate-reducing bacteria via fermentation.   Coupled with various carbon sources found within a C&D landfill (paper, adhesives, organic debris) the bacteria begin producing hydrogen sulfide according to the equation below.[4]  For every 4 grams of gypsum that decompose, 1 gram of hydrogen sulfide is produced.[5]

Equation 1

Equation 1 pic

Hydrogen sulfide (H2S) is a hazardous, colorless gas notorious for its ‘rotten egg’ odor that is detectable even at low levels.[6]  A number of instruments are available that measure toxic gas in the part per million (ppm) range, but there is a need to detect and quantify hydrogen sulfide in the much lower parts per billion (ppb) concentrations because the human odor threshold of detection of H2S is 8 ppb.[7]  To control this problem many state and local regulations limit the amount of detectable H2S in the environment, especially if a C&D landfill is nearby.  The Jerome® 605 Hydrogen Sulfide Gas Analyzer is a portable gold-film sensing instrument which is used to survey C&D sites where H2S odors are suspected.

Jerome 605

Jerome® 605 Hydrogen Sulfide Gas Analyzer

Although there are various forms of Gypsum (Anhydrous, Hemihydrate, Dihydrate,) with varying levels of purities, this paper focuses on pure synthetic gypsum dihydrate (CaSO4·2H2O).   Focusing on pure synthetic gypsum eliminates extraneous variables that may interfere with the results of this study.  Variables such as glue & paper content, variable purity samples of gypsum and non-homogenous sampling could be potential sources of error that could be analyzed in a future study.  When discussing the byproducts of gypsum it is important to address the starting concentration of the sulfate anion available to sulfate-reducing bacteria, which can be inferred from the purity value of gypsum.[4]  Although there are several wet chemical methods for determining gypsum quality, one fast and reliable method utilizes a rapid loss-on-drying instrument.[8]  Gypsum dihydrate has two levels of hydration.  One level of hydration is ‘free’ moisture which describes the water that is adsorbed to the surface of the gypsum sample.  This level of moisture may fluctuate drastically if the sample is a powder or slurry.  This free moisture will evolve at temperatures between 40 – 80°C.[9]  The second level of hydration is ‘bound’ moisture.  This moisture describes the chemical association of the dihydrate water molecules to the sulfate anion of the calcium sulfate.  This moisture level is directly proportional to the concentration of calcium sulfate and does not fluctuate based on environmental moisture.  This bound water evolves at 240°C and the resulting percent moisture content can be multiplied by 4.778 to determine the % purity of gypsum.[9]

Computrac MAX 5000XL

Computrac® MAX® 5000XL Rapid Loss-on-Drying Analyzer

The Computrac® MAX® 5000XL is a rapid loss-on-drying analyzer that is widely used in the gypsum industry to determine gypsum purity.  The MAX® 5000XL is capable of heating samples to 600°C, and can start testing at room temperature, making it an ideal candidate for testing gypsum for free and bound moisture.  Additionally, the analyzer provides real time measurements during analysis, and testing criteria can be optimized.  The MAX® 5000XL also can test for free and bound moisture simultaneously, and can output the purity of the gypsum following the test, preventing manual calculation error by technicians conducting the analysis.

The objective of this research paper is to couple both methods of analysis to observe the relationship between the purity of gypsum (calcium sulfate dihydrate) and hydrogen sulfide concentration as a sample is fermented by sulfate-reducing agents (e.g. bacteria).  The scope of this research may be beneficial for industrial hygienists concerned with C&D landfill H2S levels or for incoming gypsum processing plants who suspect poor quality gypsum. 

Methods/Results

See ‘Appendix A’ for sulfate-reducing culturing methods.

Measuring Gypsum Purity

The MAX® 5000XL has the ability to perform a slow temperature ramp allowing for different levels of hydration to evolve from a sample.  The graph shown below is an example of the pure synthetic gypsum (CaSO4·2H2O) during a temperature scan to 600°C.[10]  

Gypsum Temperature Scan

The red line demonstrates the rate (%/min) of weight loss during the temperature scan (10°C/min).  From this temperature scan, it becomes evident that the bulk of the free moisture evolves at approximately 80 °C, while the bound moisture (which is indicative of the gypsum purity) evolves at 240°C.  The MAX® 5000XL allows the user to have two separate but linked tests on the same sample.  Therefore, when pure synthetic gypsum was run in triplicate, an average of 20.84% moisture was detected from the second bound moisture peak indicating a gypsum purity of 99.6%.  This purity value does not represent a true composition of gypsum within a landfill.

In order for fermentation to occur, there must also be a carbon source.  Carbon within a C&D landfill usually comes from paper, adhesives or other organic debris mixed in with discarded wallboard.  To simulate a carbon source found in a landfill, 10% calcium citrate was added and homogenized with the 99.6% synthetic gypsum powder (30g calcium citrate, 270 g gypsum) and used as the control stock for the rest of the experiment.  Citrate is commonly used as a simple carbon source for anaerobic bacteria to utilize during fermentation.[11]

Figure 1

Bound Pic

For the ‘control’, 10 mL of sterile media A was added to 100 g of the control gypsum stock (gypsum + citrate) in a 500 mL Erlenmeyer vacuum flask.  The headspace of the vial was displaced with pure nitrogen gas for 10 minutes then securely capped.  This control vessel has all theoretical components for anaerobic fermentation of gypsum (sulfate source, carbon source, low oxygen environment) but is void of any active sulfate-reducing agents.  Two other vessels were generated from the same control stock but with 10mL of incubated ‘media A’ soil extract.

Figure 2

 % Purity

These two vessels were labeled ‘O2’ & ‘N2’ corresponding to the composition of the headspace above the inoculated slurry.  The ‘O2’ vessel contains all components for gypsum fermentation (including sulfate-reducing agents) but in atmospheric conditions (oxygen rich).  The ‘N2’ contains all components for gypsum fermentation without the presence of oxygen (nitrogen purge).   All experimental vessels were stored in the incubator at 45°C in-between testing and re-purged (if necessary) with nitrogen gas as sample was taken.[12]  Out of the ~100g sample from each vessel, ~7-8g was removed for loss-on-drying analysis on the MAX® 5000XL and run at 240 °C.

Figures 1 & 2 show the percent bound moisture and percent purity respectively for these variable conditions at day 1, 5 & 10.  It becomes clear that the ‘Pure Gypsum’ (simply powdered gypsum no citrate, no liquid media A, left in atmospheric conditions) as well as the ‘Control’ remain unchanged after 10 days.  The two vessels with active sulfate-reducing agents decrease in sulfate purity over time. The ‘N2’ is markedly lower than its oxygen counterpart in which by day 10 the percent purity drops from ~101% down to ~82%.  All tests were performed in triplicate.  Error bars represent +/- the standard deviation of each individual data set at each time point.

The results of this loss-on-drying purity assay suggest that the sulfate-reducing agents present in the soil extract are decreasing the purity of the gypsum.  Moreover, when anaerobic (N2) conditions are met, a greater rate of decomposition is observed.

Detecting hydrogen sulfide gas

As the sulfate-reducing bacteria use calcium sulfate in their metabolic cycle, hydrogen sulfide gas generated as a by-product.[11]  The Jerome® 605 was utilized to periodically measure the headspace of each vessel condition (control, O2, and N2).  This experiment was not designed to measure total H2S produced in each vessel, as the extensive sampling required to do so would potentially disrupt the incubation of the sample as well as introduce oxygen from atmospheric conditions into the vessel.

On ‘Day 1’ of incubation, 2-hour increment testing was performed by the same J605 on all three conditions during a 10-hour period.  The J605 was set to ‘auto’ range since the concentration of hydrogen sulfide was unknown.  All data points were performed in triplicate.  Figure 3 presents the with the error bars +/- one standard deviation of triplicate testing at each 2-hour time point.

Figure 3

Hydrogren Sulfide Day 1

At time point ‘0’ (when the 10 mL of media/soil extract was added to vessels), ~150 ppb of hydrogen sulfide was detected for both the O2 & N2 vessels.  This initially elevated concentration in hydrogen sulfide is most likely derived from the media A fermentation itself.  Media A contains sulfate ions intended for anaerobic growth in bacteria.  As time proceeds, a tapering effect of the O2 vessel is observed from ~150 ppb to ~48 ppb while an increase in concentration occurs with N2 vessel (~350 ppb) in the same 10 hour timespan as compared to the control vessel maintaining a level of ~3-4 ppb.

Figure 4

Hydrogen Sulfide Day 5

Figure 4 followed the same process as figure 3 but over a 5 day period in which sampling occurred once per day.  The N2 vessel (anaerobic conditions) increases from ~350 ppb on the first day to ~1600 ppb on the day 5.  Further data was collected at Day 10 not shown on Figure 4 which indicated a concentration of ~2100 ppb.  As time proceedes it appears that hydrogen sulfide production might plataeu between day 5 and 10.  Further investigation is necessary to elucidate trend.

Chart 4

Chart 4

Chart 4 presents the mean values for hydrogen sulfide gas sampling and gypsum purity for Day 1, 5, & 10.  As hydrogen sulfide gas emissions increase, gypsum purity values decrease especially under anaerobic conditions where sulfate-reducing agents are present.  Under anaerobic conditions the purity value dropped from 101.05% to 82.47%, a nearly 18.6% loss of purity.  Coupling the rapid loss-on-drying analysis from the MAX® 5000XL with the hydrogen sulfide specific Jerome® 605 demonstrates the inverse relationship between Gypsum purity and hydrogen sulfide generation due to anaerobic fermentation. 

Figure 5

Total Moisture

Figure 5 demonstrates the Total % Moisture at Day 10 (after all other analysis were performed).  These samples were poured out of their corresponding vessels (Control, O2, N2), and placed into labeled aluminum pans.  These pans were then placed in a forced air oven and dried for 1 hr at 80°C.  These samples were then cooled at room temperature in atmospheric conditions and re-pulverized into a powder and left on the counter top for 48 hours in ambient conditions.  These samples were then subjected to steadily increasing temperature from 50°C to 300°C with a temperature rate of 10°C/min.  This test shows that even if the slurry is dried and brought back to normal atmospheric conditions, the gypsum purity will remain altered after anaerobic fermentation occurs.

Discussion

Hydrogen sulfide generation due to anaerobic fermentation of gypsum is a problem in many different industries.  Construction & demolition landfills (C&D) sites in particular must monitor the hydrogen sulfide emissions from decaying gypsum-based wallboard.  Anaerobic fermentation occurs when sulfate-reducing bacteria such as the type shown below (Desulfovibrio vugaris) are present in a low oxygen environment.

Sulfate-Reducing Bacteria

Assuming these forms of bacteria have a sufficient carbon source and sulfate source (both found in wallboard), they will begin to grow and multiply.  In the process they will reduce the sulfates found in gypsum and produce hydrogen sulfide gas.  Hydrogen sulfide gas has many health risks along with having a corrosive effect on metals.  Monitoring for odor control for nearby residential areas is strictly governed by state and city regulations.  Understanding the relationship between gypsum purity and hydrogen sulfide gas is important in predicting the amount of hydrogen sulfide gas produced from fermenting gypsum.

Using a rapid loss-on-drying assay can quickly and reliably produce a measurement of gypsum quality.  This occurs because of the unique chemical association between water molecules and the sulfates of synthetic gypsum.  The Computrac® MAX® 5000XL is useful in this process because it provides a pre-linked test that removes adsorbed free moisture and subsequently tests for the bound moisture at 240°C.  Coupling this method of gypsum purity analysis with the Jerome® 605 hydrogen sulfide gas analyzer makes it possible to track the inverse trend between gypsum and H2S.

The results of this research indicate that if there is a sufficient amount of sulfate-reducing bacteria present on gypsum, then there will be a decrease in gypsum purity.  This occurs at high and low oxygen levels; however, it is dramatically increased in anaerobic environments.  Furthermore, as the gypsum purity diminishes, an increase in concentration of hydrogen sulfide is observed.  This study follows a fermentation process of 10 days; further investigation is warranted to determine the effects of longer fermentation periods.

This research was conducted under very strict laboratory controls (Temperature, oxygen environments, and pure gypsum).  These variables were selected to simulate the real-world conditions of gypsum in a C&D landfill.  Future studies may include more types of gypsum type (anhydrous and hemihydrate) as well as including real wallboard and other construction debris.  In this study, fermenting gypsum was periodically sampled for hydrogen sulfide gas concentrations.  Due to a large volume of headspace in the vial, it was impractical to quantify the total concentration of hydrogen sulfide at each time point.  Future studies may include a smaller vessel with less headspace to get a more accurate quantification of hydrogen sulfide concentration.

References

  1. Cornelis Klein & Cornelius S. Hurlbut, Jr. (1985) Manual of Mineralogy, John Wiley, 20th ed,. Pp. 352-253.
  2. “Compact Oxford English Dictionary: gypsum”
  3. http://www.gypsum.org/mediaguide.html Gypsum Association History of Gypsum Board
  4. Agency for Toxic Substances and Disease Registry.  2006. ToxFAQsTM for Hydrogen Sulfide, U.S. Department of Health and Human Services, Public Health Service.
  5. Maine Department of Health & Human Services, Maine Center for Disease Control & Prevention, Division of Environmental Health, Environmental & Occupational Health Program.  2006.  Ambient Air Guidelines for Hydrogen Sulfide.
  6. “Hydrogen Sulfide – PubChem Public Chemical Database”.  The PubChem Project.  USA: National Center for Biotechnology Information.
  7. Hydrogen Sulfide (also known as H2S, sewer gas, swamp gas, stink damp, and sour damp) Hole”.  OSHA. 12 February 2013.  Retrieved 23 July 2014.
  8. Garrett Rowe, LOD vs. Thermogravimetric Analysis. http://www.azic.com/downloads/white_papers/LOD%20vs%20Thermogravimetric%20Analysis.pdf
  9. ASTM International C471M-01 “Standard Test Methods for Chemical Analysis of Gypsum and Gypsum Products.
  10. James Moore, Moisture Analysis of Gypsum. http://www.azic.com/blog/?p=131
  11. Eric N. Kaufman et.al, A biological Process of the Reclamation of Flue Gas Desulfurization Gypsum Using Mixed Sulfate-Reducing Bacteria with Inexpensive Carbon Sources.  Applied Biochemistry and Biotechnology Volume 63-65, 1997, pp 677-693
  12. K.R. Butlin et. al., The Isolation and Cultivation of Sulphate-Reducing Bacteria. http://mic.sgmjournals.org/content/3/1/46.short
  13. American Society for Microbiology. Sulfur-Indole-Motility Agar Recipe (modified w/o indole & with Media A as broth) http://www.microbelibrary.org/library/laboratory-test/3658-motility-test-medium-protocol

Appendix A

Culturing Sulfate-Reducing Agents

Before hydrogen sulfide gas could be measured from a pure source of fermenting gypsum, a sulfate-reducing agent had to be cultured.  In this context an ‘agent’ will be synonymous with bacteria, even though there was no direct assay conducted to test for the organismal domain.  The source of the sulfate-reducing agent came from 1 cup of potting soil from an outdoor herb box.  Sample was placed in a 250 mL screw top container and approximately 50 mL of liquid ‘Media A’ was added to the potting soil making sure that there was little to no headspace.  The container was then sealed with a screw top lid and sealed with a strip of Parafilm®.  The container was then placed in the 45°C incubator overnight.[12] Having a good seal with no headspace promotes the low oxygen environment that is required for the sulfate-reducing agents to grow.  See Figure 6 below.

Figure 6

Soil Incubation

After the overnight incubation, the lid was removed and a strong ‘sulfur’ odor was noted.  The J605 was set to ‘auto’ and allowed to run above the soil suspension which yielded a reading of ~90 ppb.  Although hydrogen sulfide byproduct was detected (indicating sulfate-reducing agents), a second culturing technique was utilized to ensure sulfate-reducing agents were present.  Several Sulfur-Motility (SM) agar slants were aseptically poured from the Medium A mixed with 0.5% Agar and allowed to cool.[13]  A sterile needle was dipped into the overnight incubation soil suspension and was stabbed into the agar slant.  These agar slants were then incubated at 45°C for 48 hours.[12]  Because there is soluble iron (II) in the media (from the ferrous ammonium sulfate) any dissolved hydrogen sulfide gas produced by an organism at the site of the needle inoculation will complex with the iron and precipitate out as black iron sulfide.  The medium will change from an opaque beige color to a dark black if a sulfate-reducing agent (e.g. bacteria) is present in the media.  This media also offers insight on the agent’s level of motility due to the softness of the agar media.  See Figure 7 for schematic.

Figure 7

SM Agar Slant Inoculation

Figure 8 is a photograph of the SM agar slants.  The vial/agar slant on the left is the control vial in which a sterile needle was used (no active organism).  The vial/agar slant on the right is the vial inoculated with the suspected sulfate-reducing agents from incubated soil suspension.  The vial on the right demonstrates the presence of hydrogen sulfide production (black precipitate) as well as motility since the entire agar is black and not just at the sight on inoculation.

Figure 8

 SM Agar Slant Picture

Effective Monitoring of Hydrogen Sulfide Gas in Wastewater for Odor Control; For On-site and Laboratory Analysis

Christopher Altamirano
Lab Manager, Arizona Instrument LLC

Hydrogen sulfide (H2S) is a colorless hazardous gas known for its ‘rotten egg’ odor that many of us can detect even at low levels.[1]  Every time we pass a landfill, a wastewater facility or are brave enough to pry open the long forgotten egg salad container in the back of the fridge we have encountered H2S.  Although a number of instruments are available that measure toxic gas in the parts per million (ppm) range, there is a need to detect and measure hydrogen sulfide in the much lower parts per billion (ppb) concentrations due to odor complaints, which are traced to ppb levels.  The human odor threshold is 8 ppb and the human annoyance threshold is 40 ppb.[2]  To take control of this problem many state and local regulations limit the ppb release of hydrogen sulfide into the environment.

Wastewater treatment facilities are therefore required by law to measure and control an acceptable level of H2S exposure for their employees and surrounding civilians.  Protocols established to control and limit the odor of the H2S generated from decaying organics often include adjusting the pH, adding nitrates, or disinfecting the wastewater with chemical additives.[3]  The cost in minimizing hydrogen sulfide odor may be substantial and may place a heavy financial burden on the facility. [4] Understanding why these protocols are used for odor control as well as accurately and precisely measuring the hydrogen sulfide gas is important for ensuring safety while minimizing the use of expensive additives.  This paper briefly outlines how hydrogen sulfide is created in wastewater, how it is controlled and how you can accurately detect its presence using the Jerome® 605 Hydrogen Sulfide Analyzer (J605).  Using the J605 as an analytical tool may aid in determining the appropriate concentration of additives required to minimize H2S odor on a laboratory scale.  These empirical concentrations could then be extrapolated to full scale proportions saving the facility time, money and resources.

Jerome605

What are anaerobic bacteria and how do they contribute to H2S production?

An anaerobic bacterium is a term for a species of bacteria that is capable of functioning in an oxygen free environment.  Many of these bacteria have a distant evolutionary predecessor that thrived 3.5 billion years ago when the earth was mostly barren of oxygen but rich in sulfates.[5]  Like all forms of life, anaerobic bacteria break down sugars, lipids and proteins for energy but require some form of a chemical electron acceptor to complete its biochemical/metabolic cycle.  These sulfates (SO4-2) had become that electron acceptor (reduced) and in the process had created hydrogen sulfide gas by anaerobic fermentation.  Today, sulfate reducing bacteria such as Desulfovibrio, still play an important role in reducing sulfates in anaerobic conditions (i.e. wastewater).[5] Scanning electron micrograph shown below.

Why are anaerobic bacteria important in wastewater treatment?

Most decomposition bacteria can be categorized as chemoheterotrophic, meaning its energy source is derived from organic materials made by other organisms.[5]  Wastewater and grey water are rich with undigested organics and are the proverbial ‘all you can eat buffet’ for bacteria.  This is important in reducing the amount of solid organic waste found in wastewater.  Early in the bacterial decomposition process, aerobic (oxygen loving) bacteria dominate the feeding frenzy.  However as more and more aerobic bacteria grow and utilize the dissolved oxygen in the water, an acidic anaerobic environment is created.  This acidic, oxygen-depleted environment is now suitable for anaerobic bacteria to proliferate and consume the partially digested organic material left behind.[3]   Having an anaerobic counterpart ensures that decomposition is always occurring in any environment.

Why do pH levels, nitrates, and disinfectants affect H2S concentrations?

All forms of life function at an optimal pH.  If the pH is too high or low, proteins and lipids may denature and the organism may cease to function properly.  In the case of minimizing H2S odor, pH plays two roles.  By increasing the pH (or making the wastewater more alkaline), the anaerobic bacteria stop growing and metabolizing since their optimal pH has been altered.  Raising the pH also inhibits the release of dissolved H2S from solution, so it remains in an aqueous versus gaseous state.[4]

Nitrates (NO3-1) can also serve as a final electron acceptor in place of sulfates.  Flooding the wastewater with nitrates, forces the bacteria to utilize nitrate instead of sulfates which minimizes the H2S fermentation byproduct.[6]  Commercially available nitrates are usually in the form of liquid or powdered calcium or sodium nitrates.   Like pH, the addition of nitrates has both a biological and chemical effect on the water.  Preventative or curative attempts to minimize H2S often result in the chemical alteration of hydrogen sulfide back to ionic sulfate in the equation shown below:

H2S Nitrate reaction

The downside of adding too much nitrate is that it may cause problems down the line in purifying the water which is now spiked with excessive amounts of nitrogen.[6]  Preventative nitrate measures also tend to be a major cost in water treatment and when utilized, should only be done sparingly.

Disinfectants such as chlorine, ozone, or peroxides essentially break down the cell wall of any microbes present in the water.  These reactive compounds also combine with sulfur compounds and other hydrocarbons.[4]  These additives are usually reserved for the final step in processing wastewater but are essential for killing any harmful bacteria.  Like any other additives, these chemicals come at a cost and the addition of too much or too little have detrimental effects later in processing. 

How can you accurately monitor H2S?

Monitoring your H2S in your facilities may be as easy as walking around your work site with a hand held H2S analyzer such as the Jerome® 605.  This level of detection is important for ensuring the air quality is safe for all who may be exposed.  The J605 uses gold film technology specific for hydrogen sulfide gas with a detection limit as low as 3 ppb.  The J605 offers a continuous ‘Autosample’ mode option to monitor air quality over a period of time and saves the data which can be exported onto a spreadsheet for further analysis.

Arizona Instrument LLC (AZI) has performed several studies in which the J605 has been used not only as a safety/odor monitoring tool but as an analytical instrument in the laboratory.  Due to the volatility of hydrogen sulfide (b.p. = -60°C), one effective method for testing the concentration of dissolved H2S is to test the headspace above a liquid sample.  If the temperature and the accumulation time are well controlled, then the concentration of H2S in the headspace will be proportional to the concentration of H2S dissolved in the sample.  AZI has done extensive studies with dissolved H2S in liquid samples such as beer and wine.  A similar method can be used to detect dissolved H2S in wastewater.  The experimental design is shown below.

H2S in Beer & Wine

To run a test, an Erlenmeyer vacuum flask is connected to a Jerome® J605 Hydrogen Sulfide Analyzer by Tygon® or other suitably sized inert tubing. A liquid sample of wine or beer is poured into an Erlenmeyer flask and allowed to stir for 5 minutes. The instrument is placed in ‘Autorange’ and ‘Autosample’, sampling the headspace above the liquid sample every 2 minutes.  The instrument is then allowed to sample for 30 minutes, and the results are then summed for a total concentration of H2S.[7]

Although this particular study relates to alcoholic beverages, anaerobic fermentation still plays a key role in both processes where hydrogen sulfide gas is generated as an unwanted byproduct.  By taking in-line samples of wastewater during various stages of processing it is theoretically possible to maintain an accurate measure of hydrogen sulfide concentration on a laboratory scale.  Moreover, scaling down water treatment (addition of base, nitrates, and disinfectants) may give you better insight on quantity, the rate of reaction, and the efficiency of each of these additives may be (or any combination).

Understanding the chemistry of hydrogen sulfide and the biology of its generation is important in treating wastewater odor control.  Moreover, having a reliable and accurate instrument to measure H2S is paramount for both safety monitoring and analytical measurement. The Jerome® 605 has been shown to be a useful and versatile instrument in determining hydrogen sulfide concentrations in both air and liquid samples.  This technique may allow for more efficient bulk usage and purchasing for these odor inhibiting additives while maintaining a safe and healthy work environment in accordance with the law.

References:

[1] “Hydrogen Sulfide – PubChem Public Chemical Database”. The PubChem Project. USA: National Center for Biotechnology Information.

[2] “Hydrogen sulfide (also known as H2S, sewer gas, swamp gas, stink damp, and sour damp) Hole”. OSHA. 12 February 2013. Retrieved 22 May 2014.

[3] Mitchell, Ralph ed,. Water Pollution Microbiology, Wiley-Interscience, 1972.

[4] Palmer, Tony et. Al, Hydrogen Sulfide Control in Wastewater Collection Systems, Water & Wastes Digest, December 28, 2000.

[5] Prescott LM, Harley JP, Klein DA (1996).  Microbiology (3rd ed.). Wm. C. Brown Publishers. Pp.130-131 ISBN 0-697-29390-4.

[6] V.L Mathiodakis et. Al, Addition of Nitrate for odor control in sewer networks: Laboratory and Field Experiments.  Global NEST Journal, Vol 8, No 1, pp 37-42, 2006 Copyright© 2006 Global NESTPrinted in Greece.

[7] Moore, James et. al.  H2S in Beer and Wine.  Arizona Instrument LLC Newsletter. http://www.azic.com/downloads/white_papers/Hydrogen%20Sulfide%20in%20Beer%20and%20Wine.pdf?BLOG=Newsletter+Blog+Post&TYPE=Hydrogen+Sulfide

 

Detecting and Measuring Microbial (Unicellular Yeast) Growth on Food Products using Loss-On-Drying Moisture Analysis

by Christopher Altamirano, James Moore

Introduction

In 1825 the human population was estimated to have exceeded the 1 billion mark; a true indication of how technology and innovation was allowing us to grow in number and with longevity.[1]  Farms were becoming more industrialized and methods for preserving food and preventing spoilage became paramount for transporting food over great distances.  By the early 20th century, methods such as canning, pasteurizing and vacuum sealing were being utilized to prolong food products until they reached their destination.[2]  However these preservation methods have proven to be imperfect with many consumers suffering from food-borne illness throughout the past century.[3]  Food-borne illnesses are caused by microbial contaminants that include a vast array of parasitic, fungal, bacterial and even viral agents.  When any one of these microbial agents is consumed, the agent will replicate quickly inside the body causing a number of medical ailments and in severe cases even death.[3]

Today, we have more stringent government oversight and continuous microbial testing to ensure food safety.  These tests include selective media culturing, various forms of microscopy (or spectroscopy), enzyme linked assays, and PCR (or genetic) screenings of food samples prior to being released to the general public.[4]  However, even with the multiple detection assays at our disposal, food-borne illnesses are still a public health risk.  Reports of bacterial contamination and food recalls of chicken meat, raw spinach and pet treats still resonate in our collective consciousness and have prompted food industries and government agencies to find better ways of detecting the presence of microbial contamination.

With the exception of viruses, most food-borne microbial agents have one common life process; they eat, they grow (and/or replicate), and they produce waste.  In fact, all living things do this using a process known as respiration. [5]  Although some microbes can do this in the absence of oxygen (known as anaerobic respiration or fermentation), aerobic respiration describes the process in which energy is released from complex organic molecules (i.e. sugars, fats or proteins) in the presence of oxygen.  When this occurs gaseous carbon dioxide and water vapor are released as by-products and the organism loses mass (or weight).  The loss of mass not only comes from water vapor being released but more so by the release of carbon from a solid state into a gaseous state in the form of CO2.[5]

Cellular Respiration Equation

To test this hypothesis, we employed the use of loss-on-drying moisture analyzer (Computrac® MAX® 4000XL) with an affixed humidity air pump to keep samples at a controlled humidity.  The Computrac® MAX® 4000XL was used due to its ability to maintain a stable temperature, give accurate weight loss readings over an extended period of time, allow for downloadable graphs and data, and its internal plumbing for gas flow (humidity vapor).  Although most types of food-borne pathogens consist of bacteria (e.g. Escherichia coli, Salmonella typhi-murium, Clostridium botulinum), we, at Arizona Instrument, chose to use a common Baker’s Yeast, Saccharomyces cerevisiae as our model organism.[3]  S. cerevisiae is a relatively fast growing (90 min. doubling time) unicellular yeast that is commonly used as a leavening agent in bread and pastries.[7]  It is also innocuous in nature and NOT a human pathogen, but shares a similar physiology to some pathogenic yeasts such as Candida albicans (an opportunistic human pathogen). [7]  In this experiment, we first used an in vitro approach to demonstrate proof-of-concept by measuring weight loss on yeast inoculated potato dextrose agar plates and later tested ‘real’ food (sliced bread) using similar methods.

Potato Dextrose Agar Plates

The outcome of this investigation will offer new insights on a novel loss-on-drying approach to bioactivity measurement for food industries who wish to expand their breadth of microbial testing.  This method does not intend to replace any single traditional detection assay, but hopes to add yet another checkpoint in the quality assurance of food safety.  Because Baker’s Yeast was specifically used in this study, this approach might be particularly useful in determining the viability of certain yeast cultures for bread or for brewing industries where traditional spectroscopy driven methods (optical density) may only tell of a yeast population density, not necessarily their carbon emission capacity.

Methods

Media Preparation and Yeast Culturing

Potato Dextrose Agar (PDA)

Potato dextrose agar (PDA) is a semi-solid media which is commonly used for fungal cultivation in the lab.  Agar is a seaweed extract that has consistency of a stiff gelatin mold, but is composed of cellulose in place of proteins (i.e. gelatin). [8]  Because protein is not used as scaffolding, it can be heat sterilized at 121° C and still congeal unlike gelatin which would denature at these temperatures.  It is important to note that the agar itself is not consumed when microbes are grown on it, it is simply scaffolding that retains the liquid broth that is mix in with it (in this case PDB).

Potato Dextrose Broth (PDB)

Potato dextrose broth (PDB) is created by stewing peeled potatoes and adding dextrose (which is a simple sugar) in the correct proportions to create a nutrient broth that can be consumed by fungi (including S. Cerevisiae).  Unicellular yeasts can grow in this liquid broth in a single cell suspension and is referred to as a liquid culture. PDA or PDB can be purchased through any major biomed distributor but we chose to make it ourselves using a recipe from ‘The mushroom cultivator’ (see reference page for more details)[9]

Pouring Agar Plates

For this experiment, aluminum deep-well plates (5 inch diameter) were used with the outer rim removed (traditional petri plates made from polystyrene would also work) and sterilized at 121° C for 30 minutes.  When the molten PDA mixture came out of the oven, it was allowed to cool inside a loosely capped 500 mL Erlenmeyer flask until it reached ~50° C.  Once at this cooler temperature, ~30 mL of molten PDA was directly poured into the sterile empty plates in a sterile hood where then each of the plates were covered with a heat sterilized aluminum ‘lid’ (AZI 4KXL waffle pans) to prevent contamination.  Once the PDA solidified, the lids and plates were sealed with strips of Parafilm® to prevent excessive moisture loss and stored in a refrigerator set at 4° C.

Liquid culture of S. Cerevisiae

A portion of the PDB was withheld to cultivate a growing stock from a 7 gram packet of freeze dried Fast Rising Instant Yeast S. Cerevisiae (Bakipan®).  100 mL of PDB and ~3.5 gram of freeze dried yeast was added to a heat sterilized 500 mL beaker and mixed with a sterile loop.  The culture was periodically agitated every 30 minutes for 2 hours and either used for the experiment or stored in a refrigerator set at 4° C for later cell culturing. [9]

Inoculating PDA plates with S. Cerevisiae

Once a robust culture of yeast had been created, a flamed loop was dipped into the liquid culture and spread evenly over all of the available surface area on each plate to create a ‘lawn’ of yeast.  3 ‘loop fulls’ were used on each plate in order to supply a sufficient amount of starting population of yeast.  All inoculated plates were then inverted to prevent condensation from the lid from falling onto the lawn and disrupting cellular growth (common practice). [9] See diagram below for a visual representation.

PDA Plates

Inoculating Bread slices with S. Cerevisiae

The bread slices used for this experiment were from a Nature’s Own Honey Wheat® loaf.  The sliced bread loaf was stored in its original packaging in a refrigerator set at 4° C, but was taken out one hour prior to the experiment to allow the whole loaf to warm up to room temperature.  For the control, a slice of bread was placed onto a waffle pan and 5 drops of sterile PDB was placed onto the bread slice (4 drops being placed on the 4 corners of the bread and 1 in the center using a sterile transfer pipette).  For the experimental, the same drop placement was performed on a slice of bread but with a homogenous mixture of liquid yeast culture suspended in PDB.  (See diagram below drop placement)

Bread Slices

 

Computrac® MAX® 4000XL Programing and Procedure

Programing

In order to track weight loss in real-time, twelve 2-hour tests had to be linked to provide an accurate and continuous depiction of weight loss over a 24 hour period.  Be sure to connect an Ethernet cable to the appropriate port in the back of the instrument and enable the ‘Web Server’.  Enabling the webserver allows the analyst to download the raw data onto a Microsoft® Excel® Spreadsheet at a later point in time for data analysis.  Once you enable the optional ‘Web Server’, feature, turn the instrument on and off again to allow the instrument to recognize the new connection.  To observe the newly established instrument IP address, select ‘Menu’ à ‘Set Up Menu’à ‘Ethernet Set Up’.  Record this IP address for later data analysis (see manual for further details).

Because S. Cerevisae is not a true human pathogen, 37° C (the temperature of human body) is too warm for optimal yeast growth; therefore the temperature setting was dropped to 30° C. [7]  The purpose of using a moisture analyzer was to use it as an incubator to grow the yeast at optimal temperature and measure the weight loss over a fixed amount of time.  Test parameters for each individual 2-hour test are listed below:

Sample Size: 25.0 +/- 15.0 g

Idle Temperature: 30° C

Testing Temperature: 30° C

Pan Tare: Standard

Sample Tare: Standard

Ending Criteria: Time, 120 Minutes (2 hour maximum limit per run)

Link Test: Yes

Because each individual test will automatically read as an individual ‘% Moisture’ result, a custom equation was created to track the total percent of weight Loss.  The first 2-hour link does not require this custom equation, but the following links will in order to give an accurate real-time reading of weight loss.

Custom Equation:

((WI1-WF)/WI1)x100

With WI1 being the initial weight of the first test in the series and WF being the final weight of the current test.

Computrac® Dry Air Generator (DAG) Humidity Flow

Most food products have a substantial amount of water and because of that have a unique evaporation profile dependent on sample size, distribution, surface area and the environment it is in (humid or dry).  To minimize the evaporation for both agar plates and bread slices, a humidifier was created out of a repurposed Computrac® Dry Air Generator (DAG) with ambient air being bubbled through distilled water held in a 500 mL spouted Erlenmeyer flask, and carried past the sample chamber of the Computrac® MAX® 4000XL.  Without the modified DAG apparatus, the internal chamber of the instrument read at relative humidity of 10.6%.  After a sufficient amount of time (~30 minutes) of humid air being pumped through the chamber, the relative humidity read between 26.5% – 30.1% with the instrument holding temperature at 30°C. 

Originally, the MAX® 4000XL was designed for a nitrogen purge function in which highly volatile and potentially explosive samples could be tested in an oxygen free chamber (preventing combustion).  However, this set up was repurposed to provide a semi humid environment for the sample and for the yeast growing on it.  For further information on the Nitrogen Purge setup, consult the manual.  A diagram below shows the humidified MAX 4000XL connected by Tygon® tubing.

Tygon Tubing on the 4000XL

MAX® 4000XL Testing

Each test was started when the first link in the 12 linked series was selected and allowed to reach 30° C.  Once the temperature was attained, the analyst pushed start, the instrument tared the pan and prompted analyst to add sample within the prescribed sample window (10-40 g).  For the PDA samples (both control and inoculated alike), pans were uncovered from their original ‘lids’ and placed upside down onto the waffle pan/pan support on the 4KXL.  The instrument recognized the change in sample weight and was within the acceptable weight limits and urged analyst to close lid.  Once the lid was closed, the test began and continued for 24 hours.   

Data Analysis

Data was stored on the MAX® 4000XL instrument as well as continuously being reported to the network.  The IP address, determined at an earlier stage, was entered into the address bar of an Internet Explorer 10 Interface and the twelve linked tests were highlighted and ‘downloaded with graphs’.  This gives you the raw data in the form of an Excel® spreadsheet.  The raw data had to be reoriented by cutting and pasting into a new spreadsheet since the series was reported in the opposite order.  The ‘total % weight loss’ equation allows you to observe in real-time on the display screen but is not captured from the download; so a similar custom equation must be used in Excel® to determine the continuous % weight loss for each data point.  The equation used was as follows:

Picture1

The MAX®4000XL reports the sample weight and the rate every ~ 30 seconds.  However when a new test begins, the instrument does not immediately recognize that it is a link test and the % weight loss and rate drops back to zero.  If the first 30 second data point was left in the series the graphs produced would have drastic dips every two hours upon the onset of each test and would affect the average and therefore was omitted. 

Graphing in Excel®

The graphs presented in the Results section of this this paper were created using standard a ‘Scatter Plot’ graph in Excel® taking the averaged ‘total % weight loss’ (calculated from the previous equation) from 3 individual runs and plotted against time (in hours).  The 4 dependent variables observed were:

1)      PDA (not inoculated) – Control

2)      PDA w/ yeast inoculation

3)      Bread slice with 5 drops sterile PDB – Control

4)      Bread slice with 5 drops liquid culture

The error bars represented in each graph corresponds to the standard deviation taken at each 2-hour interval among the 3 run of each variable.

Results

PDA Weight Loss

The first hurdle in our investigation was to support the theory that ‘bioactivity’ can be measured by weight loss.  It was understood from the beginning that whatever food product was placed in the chamber would lose weight from evaporation alone.  Therefore, we needed to get a baseline evaporation curve for the nutrient rich PDA plates specifically made as an optimal media for S. Cerevisiae.  The linear ‘blue’ line of the graph represented below corresponds to three separate PDA plates void of any S. Cerivisae.

Picture2

Once the baseline rate of evaporation (more accurately the change in total % weight loss) was determined, it was predicted that if a living organism (i.e. S. Cerevisae) began consuming the dextrose in the media, that carbon dioxide and water vapor would be produced and the agar plate would begin to lose weight at a faster rate that the control plate.  Although we cannot necessarily prove that carbon dioxide or water vapors are the culprits of this loss, we can still show that a greater percentage of weight loss occurred at ~8 hrs. into the incubation as compared to the control.  As time proceeds, a higher % weight loss is observed in the experimental group.  After a 24 incubation period, it was observed that ~ 6.88% weight loss was achieved by the control, whereas 11.21% weight loss was achieved by the inoculated experimental group; a difference of ~ 4.33%.  Both variables were each performed in triplicate and averaged. Error bars represent the standard deviation at every 2 hour interval.

Bread Weight Loss

After confirming that weight loss due to bioactivity can be measured under ideal conditions, we turned our focus to a simple yet practical food item; bread.  Because yeast is used in the process of bread making, bread inherently has nutrients that yeast can readily use.  As mentioned before, each food type has a different water loss profile, and must first be determined to set a baseline to compare against weight loss due to bioactivity (or respiration).  Because bread has high moisture content and has a greater surface area than an agar plate, the rate of evaporation proved to be much greater, but eventually plateaus.  With the PDA experiment, 3 ‘loop fulls’ of liquid yeast culture was used to inoculate the experimental group which does not add a substantial amount of moisture on to the PDA plate (certainly not 4.33%).  However for the bread slice experiment, a loop of liquid culture would not be a sufficient amount of starting culture considering the surface area of all of the pores of the bread.  In order to yield a higher number of yeast onto the bread, 5 drops of liquid culture suspended in PDB was added to the bread slice (this also gives the yeast ample moisture to grow in the start).  However, 5 drops of liquid culture certainly adds a great deal of moisture to the experimental group so to even the starting moisture levels between the control and experimental, 5 drops of sterile PDB was added to the control slice (no yeast).  Over a 24 hour incubation time, the control slice of bread (blue line) losses 26.76% from its initial starting weight while the Yeast inoculated bread slice (red line) losses 28.39%; a difference of 1.63%.  This is not as big of a difference in weight loss as compared to the PDA experiment, however considering how much moisture is lost by evaporation alone, we still see a statistically significant trend between the two groups by hour 14 in a 24 hour period. Both variables were each performed in triplicate and averaged. Error bars represent the standard deviation at every 2 hour interval.

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Conclusion

Incubation based assays for determining the presence of microbial contamination on food products is not a new technique in food safety screening.  In fact, selective media culturing and spectroscopy methods often rely on growing any number of potential forms of contamination in greater number so as to be visibly perceived or detected using light spectroscopy.  Using loss-of-weight as an indication for the microbial presence on food however is a novel approach but requires a baseline evaporation curve of each product in question.  This approach allows the analyst freedom from using expensive selective media or specialized vessels for optical  density (as in some spectroscopy methods).[4]  The food product in question can be directly placed into the chamber of the MAX® 4000XL without any processing or culturing techniques. Moreover, this approach allows the analyst to view the rate of growth (via loss of weight) in real time.  It is important to note that a 24 hour incubation period (held at 30° C) was selected due to the growth profile of S. Cerevisae (doubling rate of 90 minutes).  As mentioned before, S. Cerevisiae is not normally a human pathogen which tend to be bacterial in nature.  One prime example of a food-borne bacteria is Escherichia coli (E. coli).  Under ideal conditions (37° C), E. coli has a doubling rate of only 20 minutes and if bacterial contamination is suspected a shorter incubation time may be more useful for the analyst than a 24 hour period, however some preliminary work would be required by the analyst to determine this to be true. [6]

This loss-of-weight detection assay is not a complete assay for microbial detection, but offers another method in the process of microbial determination.  This approach assumes there is a high enough microbial load already present in the food to be measured under standard incubation conditions.   This loss-of-weight approach will only detect the presence of living organisms on the food in question, it will not differentiate between innocuous and pathogenic organisms.  However, if an organism is growing on a food product at 37° C it may be a good initial indication of the presence of a potential pathogen in which other detection assays can be utilized to elucidate.

Although the repurposed Computrac® Dry Air Generator was used as a humidifier, further investigation is required to determine if this apparatus is essential in applying moisture to a growing organism, but was used as a precautionary measure to resist excessive desiccation during testing.  This brings into light the possibility of other gasses being used to fill the growth chamber.  In this experiment humidified air was primarily utilized, but humidified nitrogen was also used in a separate experiment to observe a change in % weight loss under anaerobic conditions (void of oxygen).  However, after several tests, it was determined that the % weight loss of yeast on PDA under anaerobic conditions was not statistically different to the aerobic conditions (data not shown).  This may have been caused by the strain of yeast used in this experiment, since many commercial strains have been selected for their resiliency in anaerobic conditions (i.e. within a loaf of bread).  It is interesting to note that a mild ‘alcohol’ smell was detected by analysts after these anaerobic tests had been completed.  Although this is purely speculation, it is possible for the yeast to have undergone fermentation in which carbon dioxide and ethyl alcohol may have been released instead of carbon dioxide and water.

This data may be particularly useful for members within the baking and brewing industries.  Testing the viability of yeast using other methods besides cellular enumeration might be useful since the number or density of a culture is not necessarily an indication of a strong yeast culture.  Determining the rate of growth based on % weight loss of different yeast strains might help bakers or brew masters decide on the best strain to use for their products. 

Although there may be drawbacks to having a single plate incubator (MAX® 4000XL), the data collected from the growth of an organism offers great insight on how it is interacting with your food product.  Moreover, having a stable and controlled environment means that the lid never has to be opened once the test has begun, which prevents fluctuations in humidity and temperature.  If it is in the interest of analyst, it is even possible to link a final ‘sterilization link’ at the end of the 24-hour series (depending on the sample being tested).  The MAX® 4000XL can attain temperatures as high as 275° C, though a final link set to 121° C would be sufficient to sterilize most food products (NOTE: if an inverted agar plate is being tested, it would be important for the analyst to turn the plate right side up prior to running this test, in which case a linked option would not be recommended.) 

Overall, the Computrac® MAX® 4000XL proved to be useful in detecting the presence of microbial growth based on the loss-of-weight of respiration.  This method adds one more assay for detecting potential food-borne contaminants in food and gives us insight on how real-time growth occurs on solid and semi-solid food types.

References

  1. Kennelly, John J.  Feeding 9 billion People, Ninth Annual AIA Conference, Banff March 2013, University of Alberta link http://www.albertaagrologists.ca/files/documents/5662_John_Kennelly_Feeding_9_Billion_People.pdf
  2. Tauxe R.V. Emerging foodborne diseases: an evolving public heath challenge.  Emerg Infect Dis 1997: 3:425-34
  3. World Health Organization. “Chapter 2 Foodborne Hazards in Basic Food Safety for Health Workers <internet>” (PDF). Retrieved Dec 25 2013.
  4. American Public Health Association (1992) Compendium of Methods for the Microbiological Examination of Foods 3rd Edition APHA Inc. Washington DC.  Retrieved Dec 27, 2013
  5. Stryer, Lubert(1995).  Biochemistry (fourth ed.). New York – Basingstoke:W.H. Freeman and CompanyISBN 978-0716720096
  6. Fratamico PM and Bayles DO (editor). (2005). Foodborne Pathogens: Microbiology and Biology. Caister Academic Press ISBN 978-1-904455-00-4.
  7. T. Goekhout, V. Robert, ed. (2003).  Yeasts in Food: Beneficial and Detrimental aspects. Behr’s Verlag. P. 322 ISBN 978-3-86022-961-3.
  8. “Bacterial nutrition”. Microbiology Laboratories, University of Wisconsin. Retrieved Dec 27, 2013.
  9. Stamets, P. The Mushroom Cultivator: A Practical Guide to Growing Mushrooms at Home.  http://www.ehow.com/how_5872701_make-potato-dextrose-agar.html

Comparing Traditional Oven Methods to Rapid Loss-On-Drying Methods in Powders and Bulk Solids for Moisture Analysis

Christopher Altamirano, Lab Manager
Arizona Instrument LLC

Introduction

Powders and Bulk Solids is a broadly defined genre of materials from various industries that describe a material that can be milled or processed into a powder for bulk storage or use.  For the food industry this might include milled flours, flavors or additives while the plastic industry might include resin pellets or powdered polymers.  Before final processing, it is important for these materials to be within a certain moisture range to uphold internal and external quality standards.  The moisture level of these products may change while being stored in intermediate warehouses or from being shipped in bulk around the world.  Historically, traditional oven methods (standard or vacuum) have been used to monitor the moisture levels of these products with a high degree of accuracy.  However, using these methods have proven to be quite lengthy with test times ranging from 30 minutes to several hours or even days.  No matter what the industry, test times such as these may be too long for a practical business approach in such a fast-paced economy.

A rapid loss-on-drying (LOD) Instrument for bulk solid moisture analysis uses the same theory as standard oven methods:

  1. An empty sample pan is weighed and tared
  2. Sample is added and recorded as the initial starting weight
  3. Heat is applied to sample to evolve moisture (or other volatiles)
  4. Difference in sample weight is recorded and calculated as % Moisture using the equation

With a rapid LOD instrument, the balance and heat source are coupled together allowing the user to view real-time moisture curves and rate graphs as moisture is evolved.   In the integrated system, test times are substantially faster with the same level of accuracy one would expect from traditional oven methods.

Using the Computrac® MAX® 4000XL Moisture Analyzer, various powdered samples were run in tandem with traditional oven methods to determine moisture content.  Samples included flour, food flavorings and inorganic polymers ranging as high as 14% moisture to as low as 0.03% moisture.  The instrument is robust and designed for onsite analysis using small sample sizes (<40 g).  Along with being able to view real-time graphs during each test, data from each run can also be stored via Ethernet connection or USB memory stick for graphical analysis.

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 Method

Sample Prep – All samples tested were analyzed as they were received from the manufacturer.  Samples were stored in air tight Mason jars to prevent excessive desiccation during testing. 

Test Conditions – For flour samples AOAC 925.09 or 925.10 methods were used.  For all other samples a modified 925.09 or AOAC 925.45 were used.

Computrac® MAX® 4000XL – Flour Samples

Test Temperature:  150 °C – 170 °C
Results Display:  % Moisture (3 or 4 decimals)

Computrac® MAX® 4000XL – Food Powders

Test Temperature:  85 °C – 120 °C
Results Display:  % Moisture (3 or 4 decimals)

Computrac® MAX® 4000XL – Inorganic Polymers

Test Temperature:  75 °C – 160 °C
Results Display:  % Moisture (4 decimals)

For information regarding specific parameters for each sample, contact Arizona Instrument LLC for further assistance.

All samples were sifted evenly over waffle pan and run using parameters listed above.  Flour samples that were analyzed using these methods included All-Purpose, Pastry, Whole Wheat, Soy, Black Bean, and Pinto.  Food powders samples included Vanilla, Tomato, Blueberry, Parmesan, and 3 different cheese powders.  The last set of samples included inorganic powders named polymer 1, polymer 2 and powder coating.  All trade names and manufacturer names were withheld for this study.

Results

Figure 1 represents a typical graph of whole wheat flour that can be viewed during routine testing.  The blue line corresponds to the % Moisture while the red line corresponds to the Rate (% loss per minute).  The data was downloaded into Microsoft® Excel® and graphed using graphical analytical tools in Excel®.  Once a rate drops below a set ending rate the test will terminate. (time, prediction, temp then rate are also other ending options that can be used)

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Figure 2 shows a set of flour samples analyzed with their corresponding oven method.  All oven and MAX® 4000XL tests were performed in triplicate (x3).  Error bars represent the standard deviation (S.D.) of the individual data set.

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The array of flour samples demonstrate equivalency to their corresponding oven reference methods at various moisture levels.

 It is also important to test other material to demonstrate the versatility of the MAX® 4000XL.  Figure 3a &3b are a collection of food powder flavors that also demonstrate the MAX® 4000XL’s equivalency to the oven method as well as maintaining a tight standard deviation (error bars).  Figure 3b consists of three ‘cheese powder’ samples which highlight the resolution of the instrument measuring these slightly varying moisture levels from the same product.

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Table 3 represents the raw data from ‘Cheese 1’ to demonstrate the significant difference in test times.

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Figure 4 presents another variety of samples analyzed by both standard oven and rapid LOD methods.  This set of data corresponds to the inorganic powders used in the plastic/coating industries.  The data demonstrates that even at low moisture levels, accuracy and precision are not compromised on the MAX® 4000XL as seen by the error bars (± S.D.).

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Table 4 represents the raw data with statistics of figure 4.  The MAX® 4000XL (4K) has a smaller S.D. than the oven even at low moisture levels when run in triplicate.

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Conclusion

A rapid loss-on-drying (LOD) instrument such as the Computrac® MAX® 4000XL allows the technician to accurately and precisely measure % moisture at high and low levels in various powders.  Data presented in this paper demonstrate that test times are substantially shorter when compared to standard oven references, with virtually no prep work (no need to purge pans or allow for cool time).  Having a real-time graphing LOD unit, allows the technician to view the rate of loss as its occurring or digitally save it for later analysis.  This is helpful in determining the appropriate ending criteria and maximizing the efficiency of each test.  Using the MAX® 4000XL will save both time and money and is robust enough to withstand the stresses of a distribution or manufacturing floor.

Maintaining Your Instrument: Computrac® Vapor Pro® Series

By James Moore & Chris Altamirano

Arizona Instrument LLC

Adding a new piece of equipment or specialized instrumentation to your facility has the potential to add significant value to your product.  Whether it be in the quality assurance or manufacturing process, adding a new instrument may save the company time, money, and increase the overall quality of your product.  However, adding a new method of analysis of your product requires learning new testing procedures and developing new testing parameters.  This learning curve may be the primary focus of the operator, but other tasks are important to keep in mind while adjusting to your new instrument. Tasks such as general maintenance and calibration checks should be practiced regularly to ensure the accuracy of your instrument as well as preserving the longevity for years to come.

Routine Maintenance

For operators using the moisture specific Vapor Pro ® line of instruments, routine purging of the internal tubing is recommended.  When heating samples on a daily basis within the Vapor Pro®, there is a good chance that volatilized/re-condensed material will remain deposited on the sensor of the instrument.   Making sure the sensor and tubing is void of any residue ensures accurate results.  This is completed by filling a sample vial with isopropyl alcohol (IPA) and manually moving the bottle transport in to the oven chamber.  Since IPA has a relatively low vapor pressure, it is recommended that the temperature of the instrument be set below 80 °C.  Once the transport has moved into the oven, the IPA will be pushed through the flow path allowing the fluid to flush the sensor as it passes.  Be sure the instrument is fitted with brass outlet port fitting to allow for proper drainage.  It is helpful to place an empty vial under the outlet port fitting to catch the outflow during the purge.  Leave the clean IPA vial in the instrument for approximately 30 seconds then manually move the transport out.  Once this is complete the sensor will be saturated and it could take 15-20 minutes to return to proper testing conditions.  Arizona Instrument LLC recommends this be done a minimum of once a week or as needed (depending on the type of material).  For a sample that volatilizes more readily, this type of cleaning may be needed more frequently.

Instrument Verification

When utilizing the moisture specific Vapor Pro® line of instruments, the best check to verify the accuracy of your instrument is to run a ‘RH Sensor Cal’ test.  This requires NIST (National Institute of Standards and Technology) traceable 1000µg capillary and deionized water.  To run this test, select the preprogrammed ‘RH Sensor Cal’ test and allow the instrument to heat up to 215 °C.  Gently break the surface tension of the deionized water with the capillary and water will fill the tube by capillary action.  Gently wipe any excess water droplets with your finger (Avoid cloth or tissue) and place filled capillary into a sample vial.  Seal the vial and place horizontally into the transport and begin running the test.  The test should take no more than 4-6 minutes and should yield 1000 µg ± 50 µg.  If the test results are not within this range, run a second test.  If the results are still not acceptable, call our customer service department for troubleshooting details.

Following a maintenance and calibration schedule for your instruments may seem like an extra step in your busy day, but is well worth it in the long run.  This will optimize your instrument’s performance and give you peace of mind that your instrument is working properly and accurately.  This will reduce downtime, and ensure the quality of your product.

 

 

Maintaining Your Instrument: Computrac® MAX® Series

By James Moore & Chris Altamirano

Arizona Instrument LLC

Adding a new piece of equipment or specialized instrumentation to your facility has the potential to add significant value to your product. Whether it be in the quality assurance or manufacturing process, adding a new instrument may save the company time, money, and increase the overall quality of your product.  However, adding a new method of analysis of your product requires learning new testing procedures and developing new testing parameters.  This learning curve may be the primary focus of the operator, but other tasks are important to keep in mind while adjusting to your new instrument. Tasks such as general maintenance and calibration checks should be practiced regularly to ensure the accuracy of your instrument as well as preserving the longevity for years to come.

Routine Maintenance

During the course of testing, it’s inevitable that samples will be spilled and the instrument will get dirty.  If the operator is using a Loss-on-drying instrument such as the MAX® 4000XL or MAX® 5000XL then there is increased chance of spilling sample onto the instrument due to the open chamber available to the user.  Fortunately, these instruments are designed for this type of treatment.  However, continuous build-up of material can leave the instrument looking undesirable and if the material is hazardous, it may even become dangerous for the operator.  Personal health, instrument damage and accuracy of results could be negatively affected by this build up and is therefore recommended by Arizona Instrument LLC, that the operator uses a Shop-Vac® or other nozzle based vacuum cleaner between shifts.  If the material being tested is a liquid or a gel, then the instrument should be wiped down with a cleaning agent, such as Formula 409® between shifts.  If the material is hazardous then it should be cleaned immediately using the proper procedure described on the material MSDS.

When using the MAX® 5000XL as a loss on ignition application, it is quite common for soot to build up within the test chamber.  If a significant amount of soot is observed, be sure to utilize the ‘Self Oven Cleaning’ setting.  In order to use this setting, first select ‘Self Oven Cleaning’ setting under the Calibration menu. The message on the screen will prompt the operator to remove the pan and pan support prior to beginning the program.  As soon as the pan and pan support are carefully removed from the chamber, hit start and allow the instrument to run for 45 minutes (instrument begins countdown automatically).  The instrument maintains a temperature of 550° C during this time,  which allows any residue to bake off the chamber walls.  Self-oven cleaning is recommended as needed for samples that regularly produce a full bodied plume of smoke or thick residue within the chamber.

Instrument Verification

No matter what instrument being used or the material type being tested, it is a good idea to make sure the instrument is working properly.  For the MAX® 4000XL and MAX® 5000XL, this can be performed by using a preprogrammed test labeled as ‘Weight Test’.  For this verification, the operator uses a 5g and 3 g weight.  Once the test has started, the operator places both weights on the balance as prompted and closes the lid.  The instrument will calculate the initial weight (8 g) and then begin testing.  After the test has begun, open the lid and remove the 3g weight.  Removing 3 grams from a total of 8 grams (3g + 5g) leaves you with a simulated loss on drying event of 37.50% ± 0.02%.  This is a quick verification that your instrument is measuring weight loss accurately and should ideally be performed daily to ensure accurate results.

Following a maintenance and calibration schedule for your instruments may seem like an extra step in your busy day, but is well worth it in the long run.  This will optimize your instrument’s performance and give you peace of mind that your instrument is working properly and accurately.  This will reduce downtime, and ensure the quality of your product.

Selecting the right sample size for Rapid Loss-On-Drying tests

When characterizing a material using rapid loss-on-drying technology, one of the vital parameters for accurate analysis is sample size. This is often very difficult to do because each material has unique characteristics that may influence how it should be tested. These are just some of the questions that need to be answered when determining the optimal sample size for a rapid loss-on-drying test:

Total Volatile Content – How much material is going to come off during a test? Is it 20%? Is it 2%? Is it 200ppm? The answer to this question will greatly influence the amount of material needed for an accurate test with an acceptable test time. For materials that expect losses greater than 20% only a few grams of material are needed. 5.0g ± 0.5g is a good initial testing sample size for materials with higher volatility. Once the initial analyses is finished the mass can be adjusted up or down to increase repeatability or reduce testing time, which is dependent on what condition is more desired by the user.
For materials that have a small amount of volatile content a larger sample size is needed. For materials with losses between 1 and 20% a good initial sample size is 10.0g ± 1.0g, and for materials with an expected loss less than 1% a samples size of 20.0g ± 2.0g is recommended. From these sizes the sample size can be adjusted for optimum instrument performance.

Sample Layering – Another factor to consider when selecting a sample size is sample layering. If the initial sample size selected is too large it could lead to sample material piling up. This causes the sample near the pan to be insulated from the heat source, which would cause a lower than expected result. Often times technicians attempt to compensate for this by increasing the temperature, which often leads to burning the top layer of sample material (similar to scorching the outer layer of food in an oven that is set too hot). If this is the case, try returning the testing conditions to a lower temperature and reducing the sample size while increasing surface area.

Flammability – When working with materials that can ignite, the amount of sample is a critical parameter of testing. Too much material could damage the instrument if it ignites, but too little material can hinder good repeatability from test to test. For samples that fall into this category it is best to start with 1.0g ± 0.2g of material, and increase the sample size in 0.5g increments until good testing conditions are achieved.

There are certainly other criteria that can influence the amount of sample needed, and each material will provide its own challenges for determining the optimal sample size.