Select Page
The importance of titrations in pharmaceutical analysis

The importance of titrations in pharmaceutical analysis

If you are in the pharmaceutical industry and wonder if a conversion from a manual titration to an automated titration is suitable for your work, this blog post should give you all the answers you need.

I will cover the following topics in this article (click to go directly to the topic):

Applicability of modern titration methods in pharmaceutical analysis

Perhaps you have already heard or read about automated titration and its benefits in comparison to manual titration, but are now wondering whether those guidelines are also applicable to pharmaceutical analysis.

Getting straight to the point: Yes, it is true that many USP monographs as well as USP General Chapter <541> Titrimetry still refer to the manual visual endpoint titration. But there’s good news! USP-NF General Notices and Requirements Section 6.30 states:

As long as the alternative method is fully validated and you can prove that both methods are equivalent, you are allowed to use alternative methods.

Since titration still plays an important role in pharmaceutical analytical procedures and processes, Metrohm offers a variety of applications for innumerous API monographs of the United States Pharmacopeia as well as pharmacopeia-compliant analytical instruments.

Automated titration procedure

Have you wondered about how to perform the procedure of an automated titration—how does it differ from a manual titration? Working with a pharmacopeia compliant analytical instrument from Metrohm is not so different:

 

  1. Titrant is added with an automated piston buret that safely controls the delivery of titrant to a precise level.
  2. The sample is homogenized with a stirrer.
  3. The electrode detects the titration endpoint, removing subjectivity of color changes.
  4. Results are automatically calculated and displayed allowing no room for human error.
Figure 1. Anatomy of an automatic titrator.

As shown in Figure 1, an automated titration procedure mainly consists of four steps. These steps are repeated until the end of the titration (Figure 2).

In addition, all Metrohm devices that run with proprietary tiamo® or OMNIS® software are 21 CFR Part 11 compliant meeting all ALCOA+ requirements. Thanks to improvements in productivity, accuracy, and precision, the human influence on analysis is reduced to a minimum.

Figure 2. The titration cycle illustrating the different steps in an automated titration procedure.

If you are wondering how to transfer a manual titration to automated titration, then check out our earlier blog posts on this topic. Also, download our free white paper comparing manual and automated titration.

Choice of electrodes for pharmaceutical titrations

For autotitration, either an electrode or a photometric sensor is used to detect the point of a sample analyte neutralization. Metrohm offers a wide range of different electrodes for titrations that are extremely suitable for various pharmaceutical applications. The electrode choice depends on the type of reaction, the sample, and the titrant used.

Download our free brochure to learn more.

If you want to know more about how endpoints are recognized using electrodes or photometric sensors, read our previous blog post to find out how the endpoint is determined during an autotitration.

Maybe you are not quite sure which is the best electrode for your application. Therefore, Table 1 shows an interactive electrode guide for different pharmaceutical titrations.

Type of titration Electrode Close-up view Pharma Application / API

Aqueous acid/base titrations

e.g. titrant is NaOH or HCl

phenolphthalein indicator

Combined pH electrode with reference electrolyte c(KCl) = 3 mol/L

e.g. Ecotrode Plus, Unitrode

Water-soluble acidic and basic active pharmaceutical ingredients (API) and excipients

API: Benzbromaron, Potassium carbonate, Potassium bicarbonate

Non-aqueous acid/base titrations

e.g. solvent is organic or glacial acetic acid

crystal violet indicator

Combined pH electrode with alcoholic reference electrolyte LiCl in EtOH

e.g. Solvotrode easyClean

Water-insoluble weak acids and bases

Assay of API

Acid value (free fatty acids)

API: Caffeine, Ketoconazole

Redox titrations

e.g. titrant is sodium thiosulfate

starch indicator

Pt metal electrode

e.g. combined Pt ring electrode, Pt Titrode

 

Antibiotic assays

Peroxide value in fats and oils

API: Captropril, Paracetamol, Sulfonamide

Precipitation titrations

e.g. titrant is silver nitrate

ferric ammonium sulfate indicator

Ag metal electrode

e.g. combined Ag ring electrode, Ag Titrode

Chloride content in pharmaceutical products

Iodide in oral solutions

API: Dimenhydrinate

Complexometric titrations

e.g. titrant is EDTA

hydroxy naphthol blue indicator

Ion-selective electrode

e.g. combined calcium-selective electrode with polymer membrane

Calcium content in pharmaceutical products

API: Calcium succinate

Photometric titration

e.g. titrant is EDTA

Eriochrome black T indicator

Photometric sensor

e.g. Optrode

Assay of various metal salts in APIs

API: Chondroitin sulfate, Bismuth nitrate, Zinc sulfate

Table 1. Electrode guide for pharmaceutical titrations.

To help you select the best electrode for your titrations, we have prepared a poster for you to easily find the perfect electrode for USP monographs. Additionally, you will find information about proper sensor maintenance and storage.

If you prefer, the Metrohm Electrode Finder is even easier to use. Select the reaction type and application area of your titration and we will present you with the best solution.

As documentation and traceability are critical for the pharmaceutical industry, Metrohm has developed fully digital electrodes, called «dTrodes». These dTrodes automatically store important sensor data, such as article number and serial number, calibration data and history, working life, and the calibration validity period on an integrated memory chip.

Conclusion

Metrohm is your qualified partner for all chemical and pharmaceutical analysis concerns and for analytical method validation.

In addition to full compliance with official directives, Metrohm instruments and applications comply with many of the quality control and product approval test methods cited in pharmacopoeias. Discover the solutions Metrohm offers the pharmaceutical industry (and you in particular!) for ensuring the quality and safety of your products.

Learn even more about the practical aspects of modern titration in our monograph and visit our Webinar Center for informative videos.

Need a reason to switch

from manual to automated titration?

How about FIVE?

Post written by Doris Hoffmann, Product Manager Titration at Metrohm International Headquarters, Herisau, Switzerland.

Recognition of endpoints (EP)

Recognition of endpoints (EP)

Like many of you, I gained my first practical titration experience during my chemistry studies in school. At this time, I learned how to perform a manual visual endpoint titration – and I can still remember exactly how I felt about it.

Using a manual buret filled with titrant, I added each drop individually to an Erlenmeyer flask that contained the sample solution (including the analyte to be measured) and the indicator which was added prior to the titration. With each drop and even slight color change of my sample solution, minutes passed with increasing uncertainty. I asked myself, «Have I already reached the true endpoint, should I add another drop, or have I even over-titrated?» You have probably been in the same situation yourself!

Sound familiar to you? Don’t forget to check out our other blog post about the main error sources in manual titration!

Several years have passed since then, and I am glad that I no longer have to face the challenges of performing a manual titration because Metrohm offers the possibility of automated titrations.

If you want to know how to determine the endpoint in an automated titration, I will give you all the answers you need. In the following article I will cover these topics (click to go directly to each):

    Different detection principles – an overview

    At this point you may ask yourself—if not visually, how the endpoint (EP) can be detected in an automated titration? Well, aside from the visual endpoint recognition (e.g., by a color change, the appearance of turbidity, or appearance of a precipitate), a titration EP can also be detected by the automated monitoring of a change in a chemical or physical property which occurs when the reaction is complete.

    As shown in the table below, there are many different detection principles:

    Now, let’s discuss the potentiometric and photometric EP determination in comparison to a visually recognized EP detection as they are the most commonly used determination principles for automated titrations. If you’d like to learn more about the principles of thermometric titration, read our blog post about the basics!

    Potentiometric principle

    As shown in the table above, in the potentiometric principle the concentration dependent potential (mV) of a solution is measured against a reference potential. Therefore, a silver-silver chloride (Ag/AgCl) reference electrode is used in combination with a measuring electrode (pH sensitive glass membrane or metal ring). In general, a combined sensor (electrode) including both measuring and reference electrode is used.

    Figure 1 illustrates with a simple example how a manual titration with a color change looks when being converted to an automatic system.

    Figure 1. Illustration of the same titration performed manually (left) and automatically (right).

    Step 1: Beginning of the titration before titrant is added.

    Step 2: Addition of titrant – as the titration approaches the endpoint you begin to see signs of the color change. At this point in an automatic titration the sensor will detect a change in mV signal and the titrator begins dosing the titrant in smaller volumes and at a slower rate.

    Step 3: Finally, the EP is reached with a faint pink color which corresponds with the inflection point in the titration curve.

    Step 4: Titrating beyond the endpoint leads to over titration, and here the mV signal is fairly constant.

    This is how you achieve the characteristic S-shaped titration curve you see when performing an automated titration.

    Not only acid-base titrations can be converted. Figure 2 shows how a simple chloride titration can be converted. The titrant, titrant concentration, sample size, and sample preparation remain the same.

    Figure 2. Illustration of a chloride titration – conversion from manual to automatic analysis.

    Only the indicator is replaced by the Ag Titrode, a silver ring electrode, and we get a titration curve (Figure 2, right side) with a clearly defined endpoint.

    For more examples of possible potentiometric titrations, download our free monograph «Practical Titration» or check out our Application Finder where you can find several examples for all endpoint recognition principles.

    Photometric Principle

    Titrations using color indicators are still widely used e.g. in pharmacopeias. When performed manually, the results depend, quite literally, on the eye of the beholder. Photometric titration using the Optrode makes it possible to replace this subjective determination of the equivalence point with an objective process that is completely independent of the human eye.

    The advantage here is that the chemistry does not change – that is, the standard operating procedure (SOP) generally does not have to be adapted. 

    The basis of photometric indication is the change in intensity at a particular wavelength of a light beam passing through a solution. The transmission is the primary measured variable in photometry, and is given by the light transmission (mV or % transmission) of a colored or turbid solution that is measured with a photometric sensor such as the Optrode from Metrohm.

    There are eight possible wavelengths to choose from that span nearly all color indicators used for titrations (see table below). The shaft is solvent resistant and there is no maintenance required. It connects directly to the titrator and improves accuracy and repeatability of color indicated titrations.

    I’ve also picked an example to show you how to convert an EDTA titration of manganese sulfate from manual titration to automated titration. Like in the example above, the procedure remains the same.

    Are you ready to take the leap and switch to using an automated titration system? Read our other blog post to learn more about how to transfer manual titration to autotitration.

    One advantage of automated titration is that a lower volume of chemicals is needed, resulting in less waste. With the same indicator Eriochrome Black TS, the Optrode is used at a wavelength of 610 nm. The titration curve (Figure 3, right side) shows a large potential change of the mV signal indicating a clearly defined titration endpoint.

    Figure 3. Illustration of the photometric EDTA titration of manganese sulfate according to USP.

    If you are not sure what the optimal wavelength for your titration is, then have a look at our blog post about photometric complexometric titration to learn more!

    Comparison: Optrode vs. potentiometric electrodes

    When you decide to make the switch to automated titration, there are some points to consider when comparing the Optrode with other Metrohm potentiometric electrodes. The following table lists the main criteria.

    1Optrode has a working life of tens of thousands of hours.

    You see, an autotitration is quite simple to perform and has the great advantage that a clearly defined endpoint is given.

    Believe me, whenever I`m working with such a device including a suitable electrode for an automatic titration, I have a big smile on my face thinking back to my university days: Bye bye subjectivity, time-consuming procedure, economic inefficiency and non-traceability!

    Maybe you are now also convinced to make the change in your laboratory.

    Save more money

    with automated titration

    Read our blog post to find out more.

    Post written by Doris Hoffmann, Product Manager Titration at Metrohm International Headquarters, Herisau, Switzerland.

    Multiparameter analysis in fertilizers by thermometric titration

    Multiparameter analysis in fertilizers by thermometric titration

    Agriculture without fertilizers is no longer possible – without them, today’s estimated global population of 7.9 billion people could not be supported. Fertilizers provide plants with much needed nutrients for optimal growth. The ideal fertilizer depends not only on the crop, but the soil as well. To achieve the best results, knowledge of the fertilizer composition is essential.

    To learn more about the origins of industrial fertilizers, read about the Haber-Bosch process in our series about the History of Chemistry.

    Different fertilizers for different needs

    Fertilizers can be classified in various ways, one of which being their origin. Fertilizers derived from plants and/or animals, such as dung or manure, are usually called «organic fertilizers», while fertilizers obtained from mineral salts or ores are called «inorganic fertilizers».

     The most often used classification of inorganic fertilizers is based on their nutrient composition. Classification by nutrient composition allows farmers to select the optimal fertilizer for their soil and crops. Single nutrient or straight fertilizers deliver only one nutrient. Examples are ammonium nitrate or single superphosphate. More common are multi-nutrient fertilizers consisting of two or more nutrients. Examples here include monoammonium phosphate or NPK (nitrogen-phosphate-potassium) fertilizers.

    Nutrients for plants

    The macronutrients nitrogen, phosphorus, and potassium are the main nutrients required by the plant for its growth. Other secondary nutrients such as sulfur and calcium, or micronutrients like boron are also essential but required in smaller quantities.

    Why analyze the fertilizer composition?

    Selecting the ideal fertilizer composition is essential for proper plant growth. Crops will suffer from a deficiency in nutrients, however adding an abundance of them can be detrimental, resulting in fertilizer burn for example.

    Furthermore, releasing too much fertilizers at once can lead to undesirable environmental pollution. Fertilizer producers are therefore required to specify the amount of nutrients within their products, and various norms from ISO, EN, and AOAC exist for the standardized determination of these nutrients.

    Thermometric titration for fertilizer analysis

    Traditionally the main nutrients in fertilizers are determined by analytical methods such as gravimetry, photometry, or ICP-OES. These methods require either time-consuming sample preparation or the use of expensive analysis equipment. Thermometric titration provides an inexpensive alternative solution for the analysis of potassium, phosphorus, sulfur, ammoniacal nitrogen, and urea without any time-consuming steps.

    Using thermometric titration to analyze fertilizer composition has several benefits: 

    • Analysis of multiple parameters with one device
    • Automation possibility for analyzing multiple samples a day
    • Rapid results for each parameter with titration times under 5 minutes

    Want to learn more about the analysis of fertilizers with thermometric titration? Download our free White Paper on this topic: Multiparameter analysis in fertilizers – Fast and easy via thermometric titration. 

    What is thermometric titration?

    Thermometric titration (TET) is based on the principle of enthalpy change. Each chemical reaction is associated with a change in enthalpy that in turn causes a temperature change. This temperature change during a titration can be measured with a highly sensitive thermistor in order to determine the endpoint of the titration.

    If you would like to read more about the basic principles of thermometric titration, click below for our previous  blog post «Thermometric titration – the missing piece of the puzzle».

    Metrohm’s maintenance-free Thermoprobe used for fast and reliable indication of thermometric titration endpoints.

    How are the analyses performed?

    In this section I will explain how the analyses for various macronutrients in fertilizers are done using thermometric titration.

    Thermometric titration system consisting of a 859 Titrotherm fully equipped with a Thermoprobe, titration stand and buret, and the tiamo software for the multiparameter analysis of fertilizers.
    Phosphorus

    Phosphorus is an essential macronutrient for photosynthesis and optimal crop growth, as it provides the energy to extract other nutrients from the soil. Historically, total phosphorus content is determined by gravimetric analysis. Alternatively, spectrophotometric analysis or ICP-OES may be used for the determination. These methods all require time-consuming sample preparation steps or regular calibrations.

    The thermometric titration of phosphorus is based on the gravimetric determination, but without long drying times to achieve constant weight. An appropriate aliquot of sample is added to the titration vessel and 5 mL of pH 10 buffer (ammonium chloride / ammonia) as well as 5 mL of an oxalate solution (to precipitate any interfering calcium) are added. The solution is then made up to 30 mL with deionized water and titrated with a magnesium nitrate titrant until after the exothermic endpoint.

    Figure 1. Exothermic titration curve of the phosphate determination in an NPK fertilizer (blue = titration curve, pink = second derivative showing the endpoint).

    For more detailed information about thermometric titration of phosphorus, download one of the following free application documents:

    Ammoniacal nitrogen and urea

    Nitrogen is an essential macronutrient as it is a component of amino acids (protein building blocks) and nucleic acid (building blocks of DNA). In inorganic fertilizers, nitrogen is usually present as ammonium, nitrate, or urea. Ammonia is usually determined after alkaline distillation by acid-base back-titration, while other nitrogen species are usually first converted to ammonia via digestion prior to analysis.

    With thermometric titration, a different approach is used. Ammonium ions as well as urea react exothermically with hypochlorite in a redox reaction. This reaction is further catalyzed in the presence of bromide ions in a slightly alkaline solution. 

    Figure 2. Exothermic titration curve of ammoniacal nitrogen and urea in an NPK fertilizer (blue = titration curve, pink = second derivative showing the endpoint). The first endpoint (left) corresponds to ammonia and the second one (right) to urea.

    To analyze ammoniacal nitrogen and urea, an appropriate aliquot of sample is added to the titration vessel and then 10 mL of a bromide/bicarbonate solution is added. The solution is then made up to 50 mL with deionized water and titrated with hypochlorite until after the exothermic endpoint.

    For more detailed information about thermometric titration of ammonium and urea, check out the following free application notes:

    Potassium

    Potassium is an essential macronutrient for crops, needed for regulating their water and making them more resistant to droughts. Historically, potassium content is determined by gravimetric analysis. More recently, ICP-OES is used for this determination, but the instrumentation is very expensive.

    The thermometric titration of potassium is based on the precipitation of potassium with sodium tetraphenyl borate (STPB). It is a quick titration and for this reason has already been integrated in various Chinese standards on fertilizers (HG/T 2321 for potassium dihydrogen phosphate, GB/T 20784 for potassium nitrate, and GB/T 37918 for potassium chloride).

    An appropriate aliquot of sample is added to the titration vessel. The solution is then made up to 30 mL with deionized water and titrated with STPB until after the exothermic endpoint is reached.

    Figure 3. Exothermic titration curve of the potassium determination in potash (blue = titration curve, pink = second derivative showing the endpoint).

    For more detailed information about thermometric titration of potassium, download our free application notes:

    Sulfur

    Sulfur is a secondary macronutrient and plays an important role in chloroplast growth as well as acting as a catalyst for nitrogen uptake. Sulfur is usually provided in the form of sulfate. Sulfuric acid also influences the wet phosphoric acid production process, and therefore knowledge of its content is crucial.

    Conventionally, sulfur is determined by gravimetry. The same precipitation reaction with barium is also used for the thermometric titration, without time-consuming drying to weight.

    For the analysis, an appropriate aliquot of sample is added to the titration vessel and acidified (if necessary). The solution is then made up to 30 mL with deionized water and titrated with barium chloride until after the exothermic endpoint. For improved method sensitivity, the samples can be spiked with a standard sulfuric acid solution.

    Figure 4. Exothermic titration curve of the sulfate determination in an NPK fertilizer spiked with a known amount of sulfuric acid for better endpoint recognition (blue = titration curve, pink = second derivative showing the endpoint).

    For more detailed information about thermometric titration of sulfur, download our free application documents:

    Summary

    Thermometric titration is an inexpensive analysis method without the need for costly maintenance or calibrations. It provides a rapid and robust solution for the determination of multiple parameters in fertilizers. If you wish to learn more about thermometric titration and its potential to solve application challenges do not hesitate to contact your local Metrohm representative!

    For more information, read our White Paper

    Multiparameter analysis in fertilizers – Fast and easy via thermometric titration

    Post written by Lucia Meier, Technical Editor at Metrohm International Headquarters, Herisau, Switzerland.

    Supercharge your battery research – Part 1

    Supercharge your battery research – Part 1

    Replacing traditional fuel-powered vehicles with battery-powered options is essential to reduce carbon dioxide (CO2) emissions. This greenhouse gas results from the combustion of fossil fuels, therefore limiting its input into the atmosphere will also influence global warming. Battery research therefore focuses on discovering new materials with higher energy and power density as well as a more efficient energy storage.

    Various critical parameters need to be determined to develop viable new batteries. In this first of two blog posts, I want to highlight a few of the analytical parameters which can be determined using high precision analytical instruments from Metrohm and provide some free downloads in this research area.

    What’s in a lithium battery?

    Today, lithium ion batteries are the most common rechargeable batteries available on the market. A battery consists of an anode (negative pole) and cathode (positive pole). An electrolyte facilitates charge transfer in the form of lithium ions between these two poles. Meanwhile a separator placed between anode and cathode prevents short-circuits. An example cross section can be seen in Figure 1.

    Figure 1. Cross-section illustration of a lithium ion battery. While the battery is being charged, lithium ions migrate from the cathode to the anode (from right to left), and during discharging they move from the anode to the cathode (from left to right).

    The anode is made from graphite containing intercalated lithium applied to a copper foil, while the cathode consists of metal oxides dotted with lithium ions applied to an aluminum foil. The most common transition metals used in cathode materials are cobalt, nickel, manganese, or iron. The electrolyte is an anhydrous aprotic solvent containing a lithium salt (e.g., lithium hexafluorophosphate) to facilitate charge transfer. The separator is prepared from a porous material, acting as an insulator to prevent short-circuits. The composition of all of these components has a significant influence on the battery characteristics.

    After this brief overview about the composition of a lithium battery, let’s take a look at selected key parameters and how they can be analyzed.

    Water content in battery raw materials

    Lithium-ion batteries should be free of water (concentration of H2O less than 20 mg/kg), because water reacts with the conducting salt (e.g., LiPF6) to form toxic hydrofluoric acid. Sensitive coulometric Karl Fischer titration is the ideal method for determining water content at trace levels. Water determination for solids is carried out using the Karl Fischer oven method – the residual moisture in the sample is evaporated and transferred to the titration cell where it is subsequently titrated. The working principle and advantages of the KF oven method are described in more detail in our blog post «Oven method for sample preparation in Karl Fischer titration».

    For more details on how to carry out the water determination in one of the following battery components, download our free Application Bulletin AB-434:

     

    • raw materials for the manufacture of lithium-ion batteries
    • electrode coating preparations (slurry) for anode and cathode coating
    • the coated anode and cathode foils as well as in separator foils and in packed foil layers
    • electrolytes for lithium-ion batteries

    Transition metal composition of cathode materials

    The cathode of a lithium-ion battery is usually made from metal oxides derived from cobalt, nickel, manganese, iron, or aluminum. To produce the cathode, solutions containing the desired metal salts are used. For an optimized production process, the exact content of the metals present in the solution must be known. Additionally, the metal composition within the obtained cathode material should be determined. Potentiometric titration is a suitable technique to determine the metal content in starting solutions and the finished cathode materials.

    The following mixtures of metals or metal oxides can be analyzed potentiometrically:

    • Nickel, cobalt, and manganese in solutions
    • Nickel, cobalt, and manganese in cathode materials such as cobalt tetraoxide (Co3O4), lithium manganite, or lithium cobaltite

    For more details about the potentiometric analysis of a mixture of nickel, cobalt, and manganese download our free Application Note AN-T-218.

    Analysis of lithium salts

    Potentiometric titration is also ideally suited for determining the purity of lithium salts. For lithium hydroxide and lithium carbonate, the purity is determined using an aqueous acid-base titration. It is also possible to determine carbonate impurity within lithium hydroxide using this method.

    For more details about performing the assay of lithium hydroxide and lithium carbonate, download our free Application Note AN-T-215.

    For the assay of lithium chloride and lithium nitrate, the lithium is directly titrated using the precipitation reaction between lithium and fluoride in ethanolic solutions. For more details about how to carry out the assay of lithium chloride, download Application Note AN-T-181 and for lithium nitrate download AN-T-216.

    The knowledge of other cations which might be present in lithium salts (and their concentration) is also of interest. Various cations (e.g., sodium, ammonium, or calcium) can be determined using ion chromatography (IC). IC is an efficient and precise multi-parameter method to quantify anions and cations over a wide concentration range.

    The chromatogram in Figure 2 shows the separation of lithium, sodium, and calcium in a lithium ore processing stream.

    Figure 2. Ion chromatogram of the lithium ore processing stream (1: lithium, 23.8 g/L; 2: sodium, 1.55 g/L; 3: calcium, 0.08 g/L).

    For more information on how this analysis was carried out, download our free Application Note AN-C-189.

    Eluated ions and decomposition products

    In the development and optimization of lithium-ion batteries, one of the items of special interest is the content of ions (e.g., lithium, fluoride, and hexafluorophosphate) in the electrolyte or in eluates of different components. Ion chromatography allows the determination of decomposition products in electrolyte, or anions and cations eluated for example from finished batteries. Additionally, any sample preparation steps that might be required (e.g., preconcentration, dilution, filtration) can be automated with the Metrohm Inline Sample Preparation («MISP») techniques.

    For more detailed information about selected IC applications for battery research, check out our Application Notes:

    • Cations in lithium hexafluorophosphate (AN-C-037)
    • Trace cations in lithium hexafluorophosphate (AN-CS-011)
    • Anions in electrolyte (AN-N-012)
    • Decomposition products of lithium hexaflurophosphate (AN-S-372)

    Summary

    This blog post contains only part of the analyses for battery research which are possible using Metrohm’s analytical instruments. Part 2 will deal with the electrochemical characterization of batteries and their raw materials. Don’t want to miss out? Subscribe to the blog at the bottom of the page.

    If you want to see a complete overview about the analyses which are possible with our portfolio, have a look at our brochure on Battery research and production.

    Battery research

    Positive experiences with top quality Metrohm equipment!

    Post written by Lucia Meier, Technical Editor at Metrohm International Headquarters, Herisau, Switzerland.

    Chemistry of Chocolate

    Chemistry of Chocolate

    Swiss… Belgian… Pure… Milk…

    Here we are in mid-February again, bombarded by chocolate from all sides in preparation for Valentine’s Day on the 14th. Whether in a solid bar, as a chewy truffle, or as a luxurious drink, chocolate has completely infiltrated our lives. Most people can agree that this confectionary treat is fantastic for any occasion – to be given as a gift, to recover after having a bad day, as well as to celebrate a good one – chocolate is certainly meant to be enjoyed.

    Even if you don’t like the taste, the chances are high that someone close to you does. So how can you be certain of its quality?

    Components of a chocolate bar

    For the sake of this article, let us consider the humble chocolate bar, without any extra additions (not to mention any Golden Tickets). This form can be found worldwide in nearly any grocery store or candy shop, generally designated as white, milk, or dark.

    All of this variability comes from the edible seeds in the fruit of the cacao tree, which grows in hot, tropical regions around the equator. They must be fermented and then roasted after cleaning. From this, cocoa mass is produced, which is a starting base for several uses. Cocoa butter and cocoa solids are prepared from the cocoa mass and are utilized in products ranging from foods and beverages to personal care items.

    As for chocolate bars, these are generally sweetened and modified from the pure form, which is very bitter. Milk (liquid, condensed, or powdered) is added to many types, but does not necessarily have to be present. Varying the content of the cocoa solids and cocoa butter in chocolate to different degrees results in the classifications of dark to white. While some dark chocolates do not contain any milk, white chocolates do to add to the significant amounts of cocoa fat used to produce them.

    In general, dark chocolate contains a high ratio of cocoa solids to cocoa butter and may or may not contain any milk. It may be sweetened or unsweetened. Milk chocolate is a much broader category, containing less cocoa solids but not necessarily a different cocoa butter content compared to dark chocolate, as milk fats are also introduced. Milk chocolate is also sweetened, either with sugar or other substitutes. White chocolate contains no cocoa solids at all, but a blend of cocoa butter and milk, along with sweeteners.

    Depending on the country, there are different regulations in place regarding the classification of the type of chocolate. If you are interested, you can find a selection of them here.

    What makes your favorite chocolate unique?

    Of course, more ingredients are added to chocolate bars to affect a number of things like the aroma, texture/mouthfeel, and certainly to enhance the flavor. The origin of the cacao beans, much like coffee, can impart certain characteristics to the resulting chocolate. The manufacturing process also plays a major part in determining e.g., whether the chocolate has a characteristic snap or has a distinct scent, setting it apart from other brands.

    In some cases, vegetable fats are used to replace a portion of the cocoa fats, although this may not legally be considered «chocolate» in some countries. The adjustment of long-standing recipes for certain chocolate brands has sometimes led to customer backlash, as quality is perceived to have changed. Truly, chocolate is inextricably tied to our hearts.

    Applications for chocolate quality analysis

    Nobody wants to give their Valentine a bad gift, especially out-of-date chocolate from a dubious source. Here, we have prepared some interesting analyses for different chocolate quality parameters in the laboratory.

    Sugar analysis via Ion Chromatography (IC)

    Most types of chocolate contain sugars or sugar substitutes to sweeten the underlying bitterness. Considering different regulations regarding food labeling and also nutritional content, the accurate reporting of sugars is important for manufacturers and consumers alike.

    Sugar analysis in chocolate can be performed with Metrohm IC and Pulsed Amperometric Detection (PAD). An example chromatogram of this analysis is given below.

    A small amount of commercially produced sweetened milk chocolate was weighed and dissolved into ultrapure water. After further sample preparation using Metrohm Inline Ultrafiltration, the sample (20 µL) was injected on to the Metrosep Carb 2 – 150/4.0 separation column and separated using alkaline eluent. As shown, both lactose and sucrose elute without overlap in less than 20 minutes.

    Learn more about Metrohm Inline Ultrafiltration for difficult sample matrices and safeguard your IC system!

    In this example, the sugar content was listed on the label as 47 g per 100 g portion (470 g/kg). Lactose was determined to be 94.6 g/kg, and sucrose was measured at 385.6 g/kg. To learn about what other carbohydrates, sweeteners, and more can be determined in chocolate and other foods with Metrohm IC, download our free brochure about Food Analysis and check out the table on page 25!

    Lactose content in lactose-free chocolate

    The accurate measurement of lactose in lactose-free products, including chocolate, is of special importance to consumers who are lactose-intolerant and suffer from digestive issues after eating it. Foods which are labelled as lactose-free must adhere to guidelines concerning the actual non-zero lactose content. Foodstuff containing less than 0.1 g lactose per100 g (or 100 mL) is most frequently declared as lactose-free.

    Determination of lactose in chocolate is possible with IC. Here is an chromatographic overlay of a dissolved chocolate sample with lactose spikes which was analyzed via Metrohm IC using the flexiPAD detection mode.

    Milk chocolate, labelled lactose-free measured via Metrohm IC (0.57 ± 0.06 mg/100 g lactose, n = 6).

    The sample contained 0.6 mg lactose per 100 g, with measurement of the lactose peak occurring at 13.2 minutes. The black line is the unspiked lactose-free chocolate sample, red and blue are spiked samples of increasing concentration. To prepare the samples, approximately 2.5–5 g chocolate was dissolved in heated ultrapure water, using Carrez reagents to remove excess proteins and fats from the sample matrix. Afterward, centrifugation of the samples was performed, followed by the direct injection of the supernatant (10 µL) into the IC system. Measurement was performed with the Metrosep Carb 2 – 250/4.0 separation column and an alkaline eluent.

    Interested in lactose determinations with ion chromatography? Download our free Application Notes on the Metrohm website!

    Water determination with Karl Fischer Titration

    The amount of water in foods, including chocolate, can affect their shelf life and stability, as well as contribute to other physical and chemical factors. Aside from this, during the processing stage, the amount of water present affects the flow characteristics of the chocolate mass.

    AOAC Official Method 977.10 lists Karl Fischer titration as the accepted analysis method for moisture in cacao products.

    The determination of moisture in different chocolate products is exhibited in the following downloadable poster. As an example, several samples (n = 10) of dark chocolate (45% cocoa content) were analyzed for their moisture content with Metrohm Karl Fischer titration.  Results were found to be 0.96% water with a relative standard deviation (RSD) of 2.73%. More information about this analysis can be found in our poster about automated water determination in chocolate, or in chapter 11.6 of our comprehensive Monograph about Karl Fischer titration.

    Oxidation stability with the Rancimat test

    Oxidation stability is an important quality criterion of chocolate as it provides information about the long-term stability of the product. Cocoa contains various flavonoids that act as antioxidants. Although the flavonoid content may vary amongst chocolate type, in general, the greater the content of cocoa solids in the chocolate, the greater its antioxidant effect.

    The 892 Professional Rancimat from Metrohm determines the oxidation stability of fat-containing foods and cosmetics. The Rancimat method accelerates the aging process of the sample and measures the induction time or oxidation stability index (OSI).

    Chocolate cannot be measured directly with the classical Rancimat method, as no evaluable induction time is obtained. There are many reasons for this: e.g., the fat content is too low. Traditionally, extraction of the fat from the chocolate is necessary, but not always.

    Learn more about the Rancimat method on our website, and download our free Application Note about the oxidation stability of chocolate. In this Application Note, the oxidation stability of white, milk and dark chocolate is determined without extraction.

    Cadmium in chocolate by Voltammetric analysis

    The toxic element cadmium (Cd) can be found in elevated concentrations with high bioavailability in some soils. Under such conditions, cacao trees can accumulate cadmium in the beans. Chocolate produced from the affected beans will contain elevated cadmium levels.

    Typical limit values for Cd in chocolate in the European Union are between 100 µg/kg and 800 µg/kg (EU Commission Regulation 1881/2006) depending on the cocoa content of the chocolate. Anodic stripping voltammetry (ASV) can be used to accurately determine trace quantities of cadmium in chocolate down to approximately 10 µg/kg. The method is simple to perform, specific, and free of interferences.

    Chocolate samples are first mineralized by dry ashing in a furnace at 450 °C for several hours. The remaining ash is then dissolved in an acidified matrix. The cadmium determination is then carried out on the 884 Professional VA instrument from Metrohm. To learn more about how to perform the analysis, download our free Application Note.

    Happy Valentine’s Day from us all at Metrohm!

    Post written by Dr. Alyson Lanciki, Scientific Editor at Metrohm International Headquarters, Herisau, Switzerland.