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The evolution of handheld 785 nm Raman spectroscopy: Raman extraction from fluorescence interference

The evolution of handheld 785 nm Raman spectroscopy: Raman extraction from fluorescence interference

MIRA DS (Metrohm Instant Raman Analyzer) is a handheld Raman system that identifies materials using 785 nm laser excitation. The advantages of using 785 nm Raman are well understood. Excitation with shorter wavelengths produces strong Raman scattering with short acquisition times. This results in a high signal-to-noise ratio and provides excellent spectral resolution with lower power draw. These are just some of the reasons that handheld Raman has become so popular over the last two decades.

The sensitivity of Raman at 785 nm also means that lower laser powers can be used. Lower laser powers help to protect sensitive samples from burning or ignition. The silicon detectors used at shorter wavelengths do not need to be cooled, further extending battery lifetimes. The net result is that 785 nm systems can be very small and still provide fast and accurate material identification for long hours in the field.

Learn more about how MIRA became mobile in our previous blog post.
However, while this is considered the «sweet spot» for both a strong signal and fluorescence mitigation among possible wavelengths, approximately 10% of Raman active materials fluoresce under interrogation with 785 nm Raman systems [1]. For example, Gum Arabic is a widely used filler and binding agent. When sampled with 785 nm systems, its fluorescence overwhelms the Raman signal (more on this subject later). Similarly, cutting agents (e.g., sucrose found in street drugs) fluoresce and can prevent positive identification of the target substance. Dyes can be problematic in the analysis of tablets, foodstuffs, art, and plastics as well. Often, weak Raman features can still be observed in fluorescent materials with 785 nm interrogation, but fluorescence mitigation is crucial for library matching.

Previous recommendations to overcome fluorescence

When fluorescence is an issue, 1064 nm laser excitation is often recommended. The tradeoffs include higher laser power, increased sample heating, longer interrogation times, and low Raman scattering efficiency. Often, this means larger instruments with shorter battery lifetimes. Instruments from some manufacturers require longer acquisition times that slow down sampling and can potentially damage the sample.

Is there a better way?

In a word, yes. SSE (Sequentially Shifted Excitation) can be used to remove fluorescent contributions to a Raman spectrum by using a laser that shifts the excitation wavelength as a function of the laser temperature. The result is a very large «handheld» system with a shoulder strap and a high price tag, partly due to the expensive laser used. Aside from the bulk and the cost, another issue with these systems is that the constant temperature cycling of the laser causes the system’s battery to have a short lifetime.

A Metrohm solution

Metrohm Raman has designed a fluorescence rejection system based on its compact MIRA DS package using an IPS single-mode 785 nm laser. The system is capable of producing excellent spectral resolution and flat baseline data with low laser power, short acquisition times, and all of the other excellent functionalities that users have come to expect from MIRA DS.

This fluorescence rejection system is built upon a MIRA DS platform, preserving all of its unique capabilities:

MIRA XTR DS

MIRA XTR DS is the evolution of Raman spectroscopy. It combines the smaller size, higher resolution, and lower power consumption of a 785 nm Raman instrument with patent-pending advanced algorithms to eXTRact Raman data, even from spectra that have strong fluorescence!
Figure 1. Comparison of Raman spectra of Gum Arabic powder measured by 1064 nm, 785 nm (MIRA DS), and XTR® (MIRA XTR DS).
Figure 1 contains Raman spectra from a fluorescent material, Gum Arabic powder, with traditional 785 nm and 1064 nm laser excitation, in addition to MIRA XTR DS. The improvement in resolution with XTR is obvious. Notice the very flat (uncorrected) baseline in the XTR spectrum on the bottom. This is crucial for library matching with a Pearson correlation, where the dot product between spectra and non-zero baselines contribute strongly to the correlation.

Learn more about MIRA XTR DS on our website.

Applications for MIRA XTR DS include Sensitive Site Exploitation / Intelligence Surveillance Reconnaissance (SSE/ISR) of clandestine labs and determination of synthetic routes to illicit products. MIRA XTR DS is designed for real world scenarios like the analysis of methamphetamine lab residues and identification of narcotics in street drug samples. This includes ID of narcotics, despite cutting agents that fluoresce and fail analysis at 785 nm. ORS™ combined with fluorescence rejection means that MIRA XTR DS can also delicately interrogate sensitive materials like colored explosive compounds.

Download our free White Paper below to find out more about the capabilities of MIRA XTR DS.

Classic applications improved with MIRA XTR DS

Lidocaine [2] is a local anesthetic that can also be used to cut cocaine because it enhances the immediate numbing sensation that many cocaine users associate with a high quality product. Since cocaine is typically present at only ~30% in street samples, its signal can be occluded by other components in the mixture. However, positive identification of common cutting agents like lidocaine can lead to further investigation of a suspect sample.

Traditionally, lidocaine was an issue for 785 nm Raman systems, as its fluorescence prevented both positive identification of lidocaine and detection of cocaine. MIRA XTR DS produces an excellent, fluorescence-free, resolved spectrum of lidocaine (Figure 2).

Figure 2. Comparison of Raman spectra of lidocaine hydrochloride measured by 1064 nm, 785 nm (MIRA DS), and XTR (MIRA XTR DS).
Diphenhydramine is another example of a common OTC drug that, when detected, may suggest darker dealings. It can be abused on its own, but it is also a potential precursor in the synthesis of methamphetamine. Diphenhydramine exhibits some fluorescence when interrogated with 785 nm Raman (Figure 3), but it is also typically present in mixtures with inert ingredients that fluoresce. For this type of analysis, SERS can be used to detect trace amounts of a substance. This is an excellent showcase for MIRA XTR DS, because it can perform both 785 nm Raman and SERS tests, while most 1064 nm systems currently on the market cannot be used for SERS analysis.
Figure 3. Left: MIRA XTR DS used for no-contact testing. Right: Comparison of Raman spectra of Diphenhydramine measured by 1064 nm SERS, 785 nm SERS (MIRA DS), and XTR SERS (MIRA XTR DS).
What’s the difference between Raman and SERS? Read our blog article to find out!

But MIRA XTR DS can do more!

With fluorescence mitigation, 785 nm Raman can be used more generally for material identification and chemical analyses.

Microcrystalline Cellulose

Microcrystalline cellulose (MCC) is another inert excipient that is commonly used in food production and the pharmaceutical industry. When interrogated with 785 nm Raman, its fluorescence can overwhelm the Raman signal and prevent identification and mixture matching (Figure 4).

Figure 4. Comparison of Raman spectra of MCC measured by 1064 nm, 785 nm (MIRA DS), and XTR (MIRA XTR DS).
Ketchup

Measurement of analytes in ketchup is a particularly interesting application, as it is a highly colored, complex mixture. With 785 nm testing, it shows fluorescence—with 1064 nm testing, it burns. But XTR analysis carries the added benefit of signal enhancement, returning a spectrum that clearly indicates the presence of trace lycopene in ketchup—the chemical that contributes its red color (Figure 5).

Figure 5. Comparison of Raman spectra of ketchup measured by 1064 nm, 785 nm (MIRA DS), and XTR (MIRA XTR DS).
Another important application demonstrates how MIRA XTR DS can distinguish imitation honey from the pure, unadulterated form in the pursuit of fraudulent food products, and that it shows promise for quantitative analysis. MIRA XTR DS extracts Raman spectra from materials that typically show fluorescence with 785 nm excitation, this time with sufficient resolution to detect different ratios of mixtures (Figure 6).
Figure 6. Comparison of Raman spectra of pure honey (left) and imitation honey (center) measured by 1064 nm, 785 nm (MIRA DS), and XTR (MIRA XTR DS). Right: Determination of the ratio of different mixtures of pure honey with adulterants using MIRA XTR DS. (Click image to enlarge.)

A powerful laboratory in the palm of your hand

Historically, Raman users dealt with fluorescence by using instruments with a 1064 nm laser. MIRA XTR DS combines the smaller size, higher resolution, and lower power consumption of a 785 nm laser with revolutionary machine learning to eXTRact Raman from fluorescent samples. The benefits are considerable!
  • Low power 785 nm laser interrogates sensitive samples without risk of ignition or burning.
  • Compact, pocket-sized design enables true single-handed operation of the device
  • The low power consumption means longer battery life for extended field use

MIRA XTR DS: all the best of handheld Raman with virtually unlimited applications.

Find out more about MIRA XTR DS

Download free white papers and learn more on our website.

References

[1] Christesen, S. D.; Guicheteau, J. A.; Curtiss, J. M.; Fountain, A. W. Handheld Dual-Wavelength Raman Instrument for the Detection of Chemical Agents and Explosives. Opt. Eng. 2016, 55 (7), 074103. DOI:10.1117/1.OE.55.7.074103

[2] Barat, S. A.; Abdel-Rahman, M. S. Cocaine and Lidocaine in Combination Are Synergistic Convulsants. Brain Res. 1996, 742 (1), 157–162. DOI:10.1016/S0006-8993(96)01004-9

Post written by Dr. Melissa Gelwicks, Technical Writer at Metrohm Raman, Laramie, Wyoming (USA).
Chemical analysis of sourdough: pH and total titratable acidity (TTA)

Chemical analysis of sourdough: pH and total titratable acidity (TTA)

Like many, I am fascinated by the chemistry behind baking, and in this blog I want to talk about bread—sourdough in particular. There is a well-known saying in the baking industry: «The pH value bakes and the total titratable acidity tastes». Why are these two parameters important for baking bread, and how can they be determined in the best way? This is what I want to discuss here.

A brief history of sourdough

Bread has been part of the human diet for several thousand years, although not necessarily in the forms we are familiar with today. One exception to this is sourdough bread. Wild yeast and bacteria (lactobacilli) ferment the dough naturally, creating a tangy loaf full of crevices. Despite originating in the Fertile Crescent, one of the oldest physical examples (at nearly 6,000 years old) was excavated in Switzerland, showing how widely it spread by that point already.

Currently, one of the places most well-known for its sourdough bread is San Francisco, in California. Why California? Bakers from France brought their techniques there during the Gold Rush in the mid 1800’s, and it has since become ubiquitous with the city. In fact, San Francisco has its own eponymous strain of sourdough bacteria: Fructilactobacillus sanfranciscensis.

Sourdough bread loaf full of crevices
Figure 1. Cross section of a sourdough bread loaf.

Many home bakers try to make sourdough at some point, since the ingredients are simple and no leavening agent is used, except for what nature provides. However, with so many people at home during 2020–2021, it was an ideal time for many people to see what they could produce. The development of the starter is of key importance—if there is not sufficient wild yeast and bacteria (or they do not have enough nutrients), then the dough will not rise, and you are left with a dense, chewy result. (While much has been written about how to make the best homemade sourdough, I cannot contribute to this topic, as my own baking spree focused on the Swiss Butterzopf.)

Click here to download the recipe and try it out yourself!

Lactobacilli: helpful bacteria

As their name suggests, lactobacilli produce lactic acid (Figure 2) and also acetic acid, and these give the sourdough bread its characteristic tangy, sour taste. The sourness of the bread also has positive effects on its shelf life, making it possible for our ancestors to preserve the bread for a longer time to supplement their diet.

There is another reason why the presence of this helpful bacteria is important. Without the lactic and acetic acid, it would be impossible to bake bread made from rye flour, which is commonly used in sourdough bread of northern Europe. How come?

Figure 2. Chemical structure of lactic acid.

Starch is the key component within bread and influences the shape, crumb consistency, and overall flavor. During the baking process, gelatinization occurs between the starch within the flour and the water added to the dough. However, flour also contains the enzyme amylase, which catalyzes the hydrolysis of starch into sugar. During the gelatinization process, starch is more prone to hydrolysis by amylase. Strong amylase activity at this point will have detrimental effects on the bread crumb. For wheat, the amylase is already denatured at the temperature gelatinization begins within the dough. This is not the case for rye, which gelatinizes at a lower temperature when amylase activity happens to be the highest [1]. By making an acidic (sour) dough, the amylase activity is inhibited and it becomes possible to bake bread made from rye flour.

So how much acid is necessary and when is it too much? This question brings us back to the two key parameters, pH value and total titratable acidity (TTA), I mentioned in the introduction.

Figure 3. Fermenting sourdough starter in a glass jar.

pH value regulates enzyme activity

The pH value is important to inhibit amylase in an  optimal manner. Every enzyme has an optimal pH range in which it functions the most efficiently. For amylase, the optimal pH value (highest enzyme activity) ranges from pH 5.4 to 5.8. At a lower pH value its activity will be reduced.

The pH value can be easily measured using a pH electrode. For dough analysis, an electrode such as the Spearhead electrode which can pierce into the sample is the best sensor. As the pH value is temperature dependent, the sensor measures the temperature as well.

What is the pH value?

The pH value is the negative logarithm of the hydronium concentration. Therefore, the smaller the pH value, the higher the hydronium concentration.

Pure water itself contains a small amount of free hydronium ions, and its pH value is therefore 7.

As acids release hydronium ions when they are in solution (dissociation), acidic solutions have pH values between 0 and 7. 

Contrary to this, alkaline solutions and products have even less hydronium ions than pure water. They have pH values ranging from 7 to 14. An example of an alkaline solution is lye, which is used to produce lye rolls.

For more information on pH measurement check out our other blog posts «Avoiding the most common mistakes in pH measurement» and «FAQ: All about pH calibration».

Total titratable acidity helps assess the taste

Why do we need to determine the total titratable acidity (TTA) if measuring and controlling the pH value is sufficient to regulate the amylase activity? This is because the pH value does not provide any information about the ratio of lactic acid and acetic acid present in the dough. While the amylase activity is not dependent on the ratio of the two, the composition is important for the taste. For optimal sourdough flavor, the ratio of lactic acid to acetic acid should lie between 3:1 and 4:1. If the ratio shifts towards containing more acetic acid, the taste usually becomes too sour.

Weaker acids such as lactic acid and acetic acid do not completely dissociate, meaning not all acid molecules present will release their hydrogen ion. As lactic acid is a stronger acid in comparison to acetic acid, more lactic acid will dissociate and thus contribute more to the pH value. By determining the TTA, it is possible to find out what the total amount of acids is within the dough.

For the determination of the TTA, dough is homogenized with water to obtain a suspension. It is then titrated to a pH value of 8.5 with 0.1 molar sodium hydroxide solution. The use of an automated titrator provides reliable results without human interference (Figure 4).

Figure 4. A robust and compact titrator for the determination of the total titratable acidity

Before the titration starts, the pH value of the suspension can be determined easily, so you get both parameters (pH value and TTA) without needing to do double the work. For more detailed information on how the analysis is done, download our free Application Note.

pH value and TTA for the perfect sourdough quality

By combining the information about the pH value and TTA it becomes possible to assess the quality of sourdough and thus maintain a constant quality in the final product, especially if delays in the production process occur.

It also becomes possible to detect changes in the sourdough starter which might occur if storage conditions cannot be maintained, and thus provide critical information when it is time to prepare a new starter.

Table 1. Common pH and TTA values for various kind of breads [1].
Bread type pH value TTA
Wheat bread 5.4–6.0 4–6
Wheat mixed bread 5.0–5.3 6–8
Rye mixed bread 4.5–4.8 7–9
Rye bread 4.3–4.7 8–10
Rye bread (coarsely ground) 4.2–4.6 9–14

Lessons learned

I hope this blog post on the chemistry of sourdough has given you some new insights on this fascinating kind of bread.

As for myself, I will probably not venture into the sourdough baking arena but stick with my homemade Butterzopf.

Figure 5Butterzopf (made by the author): a traditional Swiss bread usually consumed on Sundays for brunch.

If you are interested in other blog articles related to yeast, check out our post about beer brewing: «Making a better beer with chemistry». If you have more of a sweet tooth then read our blog post on the «Chemistry of chocolate».

The chemistry of bread

Straightforward determination of pH value and total titratable acidity (TTA) in dough

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

Analysis of prebiotics with IC-PAD: Improving AOAC 2001.02

Analysis of prebiotics with IC-PAD: Improving AOAC 2001.02

Our diet is critical for our health. In the past several years, interest has increased in food additives and dietary supplements such as prebiotics like β-galactooligosaccharides (GOSs). The determination of total GOS contents in food and supplements is essential to fulfill strict food labeling and safety requirements. The most widely used method for total GOS determination is based on enzymatic hydrolysis to break down the complex molecules into simple carbohydrates prior to their chromatographic analysis. This article outlines the advantage of using an improvement to AOAC Method 2001.02 using ion chromatography with amperometric detection (IC-PAD) and full sample automation after enzymatic hydrolysis.

What are GOSs?

GOSs are chains of galactose units with an optional glucose end. They are often naturally present in small amounts in various foods and beverages.

Initially discovered as major constituents of human breast milk (present up to 12 g/L), GOSs are added as a prebiotic supplement to infant formulas. They show bifidogenic effects, meaning they support growth and well-being of non-pathogenic gut bacteria.

GOS supplements are available either raw, or as concentrated powders or syrups, and are subsequently used by food manufacturers to enrich consumer products or sold as supplements.

GOS labeling requirements

The ongoing growth of global prebiotic and GOS markets is a result of increasing consumer awareness regarding healthy eating. Similarly, increased demand regarding food quality has led to stricter, more comprehensive rules for food labeling and safety (e.g., EU 1169/2011 and  EU 2015/2283). The determination of total GOS contents in food, supplements, or raw products is thus essential to fulfill such requirements.

Studies about GOS health effects recommend maximum doses under 30 g per day, though this is much stricter for infant formulas. Otherwise, there are no other limits regarding GOS content in food or as nutritional supplements.

AOAC 2001.02

The most widely used method to measure total GOSs in food products is the standard method AOAC 2001.02. This method is based on the extraction of GOS from a sample followed by enzymatic hydrolysis of the oligosaccharides into monosaccharides and their subsequent analyses with high performance anion exchange chromatography with pulsed amperometric detection.

Figure 1. Schematic for determination of total GOS contents using ion chromatography with pulsed amperometric detection (IC-PAD) according to AOAC 2001.02, and an optimized method from Metrohm (in green). Chromatography for anions in AOAC is referred as HPAEC (high performance anion exchange chromatography) but is simplified here to the generic term of IC.

In AOAC, chromatography for anions is referred to as HPAEC (high performance anion exchange chromatography) but here we will simplify this to the generic term of IC.

The key to AOAC 2001.02 is the comparison of a control solution with one which has been treated and hydrolyzed with an enzyme (β-galactosidase). The enzyme catalyzes the splitting of glycosidic bonds and hydrolyzes GOSs and lactose into glucose and galactose. The concentration differences of free galactose and lactose determined in these two solutions is used to calculate the total GOSs (Figure 1).

Improvements to the AOAC Method

The sample preparation for AOAC 2001.02 is rather complex: one shortcoming is the incubation of the reference solution with the deactivated enzyme (which is rather expensive) to determine the initial carbohydrate concentrations (Figure 1) rather than using the pure extract. Another critical point is the sample dilution procedure, which is supposed to be done in acetonitrile, while standards are based on ultrapure water.

Here, the focus was to simplify the entire procedure to increase the ease of use and the overall efficiency of the method.

The improved method for total GOS content analysis uses the extract for measuring of the initial glucose, galactose, and lactose concentrations (Figure 1 Assay 1). However, the deactivated enzyme was not used, and instead comparisons were made to see if its presence had any effect on the results. This step was eliminated after proving results equivalent to AOAC 2001.02 Assay 1 (with the deactivated enzyme), but chemical expenses and additional manual work are reduced. The total GOS content is therefore calculated from the analyte concentrations in Assay 1 (without any enzyme) and Assay 2 (extract with the active enzyme) (Figure 2).

Figure 2. Overlaid chromatograms of Bimuno (prebiotic supplement), untreated (black) and treated with enzyme (orange).

Want to know more details about the application? Download our free Application Note AN-P-087 about total GOS analysis in foods with ion chromatography!

Aside from the enzyme usage, the official AOAC method for analysis of total GOSs suggests that standards be prepared in ultrapure water (UPW) while samples are to be diluted with 20% acetonitrile. A control experiment was performed to compare results between:

  • Dilutions in UPW evaluated with UPW calibration (“UPW option”)
  • Dilutions in acetonitrile evaluated with UPW calibration (AOAC 2001.02)
  • Dilutions in acetonitrile evaluated with acetonitrile calibration (“ACN option”)

Reproducibility of total GOS contents was compared among the three options, with the UPW and AOAC preparation options exhibiting similar results. The ACN option resulted in lower total GOS contents than the others. Additionally, the acetonitrile did not seem to lend a stabilizing effect to the samples. This supports the improvement of the AOAC method by performing sample dilutions with UPW instead of acetonitrile, saving unnecessary reagents and limiting the chemical imprint of the analysis.

Results

Overall, the satisfying variability, target and spike recoveries (Application Note AN-P-087), together with the interference tests proved the modified method as valuable and robust. With limits of detection (LODs) of 0.1 mg/L (galactose) and 0.2 mg/L (glucose, lactose) in solution, even low total GOS contents can be determined with high precision.

Summary

As a multicomponent method, ion chromatography with amperometric detection is a very selective, sensitive, and robust analysis method for carbohydrates without any additional derivatization steps. In combination with enzymatic treatment, even more complex carbohydrates can be quantified.

This research presents an update to the standard AOAC method for total GOS determination in foodstuffs. With the same principle (enzymatic hydrolysis of complex GOS molecules followed by chromatographic analysis of simple carbohydrates), analytical method efficiency was improved in favor of laboratory time and running costs. Additional automation steps (e.g., Metrohm Inline Dilution and automatic calibrations) can further improve the method efficiency.

Want more information about the simplified method for total GOSs via IC-PAD? More details about the improvement of AOAC method 2001.02 by reducing manual laboratory work and eliminating expensive reagents can be found in our article published in The Column from LC/GC (2021): Improving on AOAC 2001.02: GOS Determination in Foods Using HPAEC–PAD.

Read our article in LC/GC The Column (2021)

Improving on AOAC 2001.02: GOS Determination in Foods Using HPAEC–PAD

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

Recipes with Raman

Recipes with Raman

Many of us have spent more time in the kitchen in the past year than usual, (re)discovering our culinary skills with varying degrees of success. Our pantries have been kept full, and our stoves on for a year (and counting) since our normal, social ways of life have been curtailed by home office regulations, online schooling, and the sweeping closures of bars and restaurants.

Cooking at home can mean a number of things. Some people rely on «Chef Mike» (i.e., the microwave) to prepare their meals, while others turn humble ingredients into haute cuisine dishes. However, most people would probably agree that the keys to delicious and nutritious meals are fresh, high quality ingredients.

What is on your menu today? For breakfast, perhaps toast and some fresh pressed orange juice, lunch is maybe a quiche with tomatoes and cheese, and for dinner, stir-fried vegetables accompanied by a glass of good wine. Hungry yet?

With all of this talk about food, how can you be certain that the ingredients you are using in the kitchen are of the highest quality? You may trust in the grocery store, the brand, or the farmer at your local market, but do you know how different food quality parameters are measured?

One technique provides rapid, non-destructive and specific food quality testing: Raman spectroscopy. Whether you are looking to determine the ripeness of fruits or vegetables, the adulteration of spices or dairy products, or contamination of foods with banned pesticides, Raman spectroscopy is at the cutting edge of food quality analysis.

If you want to refresh your knowledge about Raman spectroscopy, have a look at our previous blog post about Mira, which includes some history about the technique.

To learn more about the analysis of trace adulterants in foods and beverages, read our blog post all about measurement with SERS (surface‐enhanced Raman scattering).

Are you confused about the differences between Raman spectroscopy and SERS? You’re not alone! Check out our blog post about these two techniques and learn about their benefits.

Here, we share a selection of peer-reviewed articles from the scientific community using Raman spectroscopy and portable instrumentation from B&W Tek, a Metrohm Group Company and Metrohm Raman to address quality issues of food. Enjoy your meal! Bon appetit!

~~ Starter ~~

To begin, maybe you would be interested in sharing a bottle of red wine with your companion as you snack on some crispy bread sticks. Red wines are made from red varieties of grapes, whose color is imparted through the crushing process as the skins soak in the sugary juices. Phenolic compounds derived from the grape skins can be beneficial to human health, and can be determined with Raman spectroscopy [1].

It’s not only beneficial compounds but also harmful contaminants that can be measured in beverages with Raman spectroscopy. Fungicides can also be detected in wine with SERS. Download our free Application Note if you want to find out more.

Watch our video below to see how methanol in alcoholic drinks is quantified rapidly without sample preparation – right at the bottle!

Snacking on prepackaged foods when you are on the go, or when you don’t feel like cooking at the moment, is something we have all done. The moisture levels in most of these foods is kept to a minimum, especially in those meant to have long shelf lives. Water content above certain levels allows harmful bacteria to grow, which is one of the major reasons to always consult the date of packaged foods before consumption. Eating contaminated foods can cause severe sickness and even death. It is possible to determine whether such low moisture foods (LMFs) contain harmful levels of these bacteria with SERS [2].

What else do both of these applications have in common? Both of them utilize the portable i-Raman Plus instrument from B&W Tek. For more information, download our free application note: Portable Raman for Quantification of Methanol in Contaminated Spirits.

~~Main Course~~

Depending on what you are in the mood for, anything is possible. Some tomatoes, vegetables, spices, perhaps meat (if you eat it) and a starch are on the menu today, ready to be turned into almost any dish.

Determining whether fresh foods are at peak ripeness can be a tricky process, not necessarily just the change of a color. The ripeness of a fruit or vegetable indicates its antioxidant content, as well as nutrients and other beneficial compounds. Monitoring the ripening process is possible with portable Raman spectroscopy [3], such as the B&W Tek i-Raman Pro.

Some of us like a little heat in our meals. Unfortunately, the adulteration of spices like chili powder (sometimes known as cayenne powder) is common, as cheap and harmful coloring agents are added to achieve more profits at the cost of human health. These synthetic dyes are able to be determined easily even at trace levels with SERS [4].

Download our free Application Note to learn more about the detection of trace levels of Rhodamine B in cayenne powder with SERS.

Some types of cheese command a high price for what seems like just a small pinch. One such type is Parmigiano Reggiano, an Italian cheese with a protected denomination of origin (PDO) quality marker, made in compliance with several production rules. These cheeses are subject to counterfeiting, but luckily this is easy to determine on-site without damaging the sample using handheld Raman spectroscopy [5].

The price of meat varies according to several reasons, even for the same animal source, section (cut), and portion size. Among these is the origin of the meat, as well as how it was produced (e.g., organic or a factory farm). Determining the difference between premium meat products and lower quality ones is possible with handheld Raman systems [6] such as Mira from Metrohm Raman. Not only these differences but also the freshness of meat during the production process can be measured with portable Raman devices [7] like the i-Raman Plus from B&W Tek.

Using lower quality cooking oil with a low smoke point at high temperatures can result in consumption of harmful byproducts formed during cooking. Older oils have a lower antioxidant content as a result of the aging process, and can become rancid when the antioxidant properties vanish. For these reasons, high quality edible oils full of antioxidants are worth much more, but are also susceptible to adulteration with cheaper ingredients. It is possible to not only determine the purity of edible oils by Raman spectroscopy [8] but also the heat stability of different types of oils [9].

For more information about the analysis of edible oils by Raman spectroscopy, download our free Application Notes and our White Paper below!

~~ Dessert ~~

After dinner is over, a hot beverage like tea can be nice to cleanse the palate. How can you be sure that the tea is free of banned pesticides, other than buying from a trusted organic label? SERS allows rapid identification of such substances in tea leaves [10].

To learn more about detecting illegal compounds such as herbicides on tea leaves, download our free Application Note.

The honey you put in your tea or drizzle over your dessert can also be subjected to tampering. Depending on the type of flower or the origin of the honey, costs can vary widely for the same volume. Some honeys (e.g., Manuka) claim to impart certain health benefits, and therefore many lower quality products with cheap sweeteners (e.g., high fructose corn syrup) are falsely labeled as such and sold at a higher price point to unsuspecting consumers. It is possible to detect honey adulteration [11] and even its botanical origin [12] with Raman spectroscopy.

Not only tea and honey, but also coffee and the milk added to it can be analyzed with Raman spectroscopy to determine various quality markers and adulterants.

The protein content of milk can be falsely enhanced with the addition of melamine. This compound is now monitored in dairy products due to scandals which led to deaths from kidney damage. Melamine [13] and other substances which can contribute to ill health effects [14] can be easily determined in milk with SERS.

Want to learn more about Melamine and how to measure it with SERS? Check out our free Application Note for further information.

Download our free Application Note to learn about the rapid detection of the alkaloid trigonelline in coffee, which reduces in concentration the darker the beans are roasted.

The ripeness of fruits and vegetables is not just important information when planning meals, but it is also critical for food transport. Perishable fruits and vegetables are often shipped in an unripened state so they arrive at their destination in top condition.

Freshness in citrus fruits can be determined with portable Raman instruments by measuring the carotenoid content [15].

Aside from the freshness, it is also possible to detect if pesticides, fungicides, herbicides or other harmful substances have been sprayed onto fruits using SERS [16].

Check out our selection of free Application Notes below about the determination of these kinds of substances on different fruits with Misa.

Several food quality parameters can be measured quickly and easily with Raman spectroscopy without the need to open bottles or destroy samples. Portable and handheld instruments make measurements simple to perform nearly anywhere. Visit the Metrohm website to learn more about the possibilities with Raman!

Learn more about rapid food analysis with Raman spectroscopy

Download free applications directly from our website.

References

[1] Dranca, F.; Oroian, M. Kinetic Improvement of Bioactive Compounds Extraction from Red Grape (Vitis vinifera Moldova) Pomace by Ultrasonic Treatment. Foods 2019, 8, 353. doi:10.3390/foods8080353

[2] Pan, C.; Zhu, B.; Yu, C. A Dual Immunological Raman-Enabled Crosschecking Test (DIRECT) for Detection of Bacteria in Low Moisture Food. Biosensors 2020, 10, 200. doi:10.3390/bios10120200

[3] Trebolazabala, J.; Maguregui, M.; Morillas, H.; et al. Portable Raman spectroscopy for an in-situ monitoring the ripening of tomato (Solanum lycopersicum) fruits. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2017, 180, 138–143. doi:10.1016/j.saa.2017.03.024

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Post written by Dr. Sara Seiffert (Product Specialist Spectroscopy at Metrohm Deutschland) and Dr. Alyson Lanciki (Scientific Editor at Metrohm International Headquarters).

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.