Lab Course

Stable Isotope Analysis for FoodAuthenticity Control

Prof. Maik A.Jochmann

Submitted by:

Group 3Kumari Soni ES0300951500Mili Shah ES0300990600

Contents

1. Introduction............................................................................................... 12. Materials and method............................................................................................................72.1 Materials............................................................................................................................. 7 2.2 Instrument and tools............ ......................................................................................... 8 Procedure........................................... .............................................................................. 93. Results....................................................................................... .............................................105. Discussion..............................................................................................................................156. Questions................................................................................................................................177. Conclusion..................................................................................................................... 198. References..............................................................................................................................19

INTRODUCTION

The term food authenticity refers to whether the food purchased by a consumer matches its description and Food control must be able to verify its description. Authenticity and origin of food products is a major task in quality control for consumer protection in a globalized world with increasing competition on the food markets. Artificial ingredients used for adulteration can show chemical identity to the natural ingredients. In such cases, there is no health concern involved but consumer fraud is conducted by mislabeling. Conventional methods applying GC-MS or HPLC-MS fail because they are not able to distinguish chemically identical compounds and thus differentiate between natural and synthetic origin or provenances of valuable ingredients. In contrast, stable isotope ratio analysis of light elements such as carbon, hydrogen, nitrogen and oxygen Isotopeis nowadays a widely used method to answer questions about food adulteration and geographical origin tracking [Lab manual 2014 ]. Ratio Mass Spectrometry is today one of the Standard technique for authencity control of food and pharmaceutical products. These techniques are used to identify the differences in the isotopic signatures of the samples. Such as in plants, carbon is primarily the cosequences of three photosynthesis pathways(C3,C4 and CAM). C3 plants has a more negative 13C value than those of C4 plants . [1 ,2]

C3 plantsC4 plantsCAM plants

Cereals: Wheat, Barley, Oat, Rye, RiceSugar canePineapple

Sugar BeetMaizeVanilla

PotatoMilletAgave

GrapeSorghumCacti/Succulents

Citrus fruitSedgeOrchid

Cotton

Trees (including maple, olives)

Groundnut/Peanut, Coconut

All Cruciferae including rape

Soybean

Sunflower

Cocoa

Sesame

Most vegetables

Table 1 Classification of plants with regard to carbon dioxide fixation pathway.[2]

Generally in the case of BSIA (Bulk Stable Isotope Analysis),EA-IRMS are used where the compounds are converted to low molecular weight gases (CO2, N2, CO, SO2 and H2) before separation by packed column gas chromatography whereas in CSIA(Compound Specific Stable Isotope analysis), compounds of complex mixtures are separated by chromatography (GC, LC) as preliminary step into a low molecular weight gases before subsequent introduction to IRMS. Thus, CSIA allows a differentiation of isotope ratios of individual compounds, but the isotope ratio of only one element at the same time can be measured for the separated compounds.In this practical course, EA-IRMS was applied for the authenticity control of sugar.

Figure 1 Difference between bulk stable isotope and compound-specific stable isotope analysis

In our experiment , the main goal is to lean the principles behind EA-IRMS as well how to prepare ,measure and evaluate isotope values.Comparison of different sugars reveals the difference between processed and natural sugars.

EA-IRMSAmongst the several peripheral devices that have been so far coupled to continuous flow- IRMS, the most prominent one is EA-IRMS in stable isotopes analysis.Modern commercially available elemental analyzer (EA) provides an automated means for on-line high-precision isotope ratios for bulk analysis of solid and nonvolatile liquid samples.In case of BSIA, the compounds are converted to low molecular weight gases (CO2, N2, CO, SO2 and H2) (the content of sulfur can be ignored in sugar) before analyzed.Depending on the type of sample, different preparations such as grinding, dry freezing, and aliquotion needed to be carried out prior to measurement by EA-IRMS. The prepared samples are weighed then filled in a tin capsule, and then are loaded into a carousel in the autosampler, from which they are introduced into the conversion reactor.

Figure. 2 The scheme shows the setup of an elemental analyzer in series with an interface and IRMS for nitrogen and carbon isotope ratios analysis. Sources by script of lab course-Stable Isotope Analysis for Food Authenticity Control (Uni Duisburg-Essen SS2014)

In figure 2 above, the typical setup of an EA-IRMS for carbon and nitrogen isotope ratio measurements is shown. After the introduction, the sample fall into a heated reactor that contains oxidation catalyst, where combustion takes place in a He atmosphere with an excess of oxygen. Quartz wool is used here for holding the reactor temperature at approximately 1000 C, at which the sample is completely converted to CO2, N2, O2 and water.Transportation of Combustion products is enhanced by flowing He through a reduction furnace filled with copper and is heated at around 600 C for the removal of excess oxygen and conversion of nitrous oxides into elemental nitrogen(N2). Without removal of NOx gases, the 15N2 will be confused and could be misread in the measurement.Drying trap is used to remove any excess of water in the system. And the gas-phase products are separated by GC column incorporated into the elemental analyzer, which is maintained isothermally at 40 C. The separated gases are transported into IRMS via dual-inlet interface with reference gas. Due to the misbalance of high C/N ratios of most organic materials, very small N2 signals are measured along with large CO2. A dilutor, therefore, is added, which dilutes the sample gases by adding helium for the adjustment of the larger CO2 peak. This would result in obtaining the similar precision with very different C and N amounts in the sample.General assembly of IRMS All the types of mass spectrometers are used to measure the isotope abundances but not with desired precision.For obtaining high-precision and accurate measurements of the light elements(C,H,N,O) isotope ratio mass spectrometers (IRMS) is commonly used. There are generally three parts i)the ion source ii)the mass analyzer iii) the ion collection assembly. For IRMS analysis analytes are converted in the form of limited number of gases, which should be isotopically representative of the original sample.IRMS usually used to detect the 13C/12C, 2H/1H, 15N/14N, 18O/16O, and 34S/32S ratios but sample should enter the IRMS as gas and then we can analyse the pure gases of carbon dioxide(CO2), hydrogen(H2 ) , Nitrogen(N2), Oxygen(O), and sulphur dioxide (SO2 ), which are usually carried by He gas.Figure 3: Schematic representation of IRMS.The precision for light elements are obtained only by gas source magnetic sector field instruments where the simultaneous measurement of gasoeous species (ionized) occur in array of Faraday collector .The magnetic sector separates the charged molecules by mass. Lighter gases such as hydrogenH2) are trapped at first in the magnetic sector, and higher mass gases such as CO2 and N2 will be captured in the cups in universal collectors.

Figure 4. Diagram of a generic isotope ratio mass spectrometer, without including inlets.

To increase the ionization probability, IRMS employs an electron impact (EI) ion source .As the analyte stream passes through the electron beam, the analyte molecules are either ionized or pass out of the source, then pumped away. Ionized molecules are then rapidly accelerated into the analyzer section before collisions can take place. The analyzer of IRMS is always a single magnetic sector as shown in figure 4. Stability and Transmission are optimized by mass resolution which is its one of the characteristic feature. The IRMS analyzer section is designed with a proper resolution which is sufficient enough to separate the masses of interest under continuous maximum transmission. Sometimes, the analyzer has a permanent magnet, which means the behaviors of molecules could not be changed by the analyzer itself. Hence, the accelerating voltage is required for the appropriate adjustments.

The major reason for keeping Faraday cups(FC) is that ,at the exact same interval of time the multiple ion currents are measured and hence the fluctuations in ion sources is completely cancelled also peak jumping and settling times are avoided. Each cup is used for trapping one kind of interested ion beam. The collector system is aligned so that the ions which exists the magnetic sector field analyzer is striked to FC collector electrodes which are positioned along the focal plane of Mass Spectrometer.

Figure 5: Schematic depicting of a triple-collector IRMS system to analyze the isotopic composition of CO2. The same principle is used for analysis of the isotopes of 16O, just with a different set-up of the collectors. (Centre for ice and climate ,Neils Bohr Institute http://www.iceandclimate.nbi.ku.dk/research/drill_analysing/cutting_and_analysing_ice_cores/isotope_measurement/mass_spectrometry/)The dual-inlet systems are also one of the necessary part of modern IRMS, which are controlled by computer and enable the storage of sample and standard gas. Furthermore, with the help of automated valves, the sample and standard gases can be alternately delivered in the IRMS or a waste line vacuum pump. It is also worth to mention that the connection between the gas reservoirs and the valve is made with thin capillaries which provide the pressure for different sample gases. As a result, the dual-inlet systems are responsible for the highest precision of a non-transient signal. The flow through the capillaries is viscous, which prevents the isotopically enriched gas from diffusing back into the reservoirs. Therefore, a transient signal is induced by continuous flow system. And this viscous flow condition enables lower limit to the minimumamount of gas to be determined with higher precision.

2. MATERIALS AND METHODS2.1. Materials- Sugars of different origin (8 samples)-Acetanilide (Internal laboratory standard for elemental analysis) purchased from Merck(Darmstadt, Germany)-Helium 5.0 (Air Liquide, Duesseldorf, Germany)2.2. Instruments- Tin capsules, tweezers, mortar, micro-well plate and micro-spatulas- A fine balance from Sartorius SE2 (Hamburg, Germany) - for weighing of sample and standard- A vario PYRO CUBE (Elementar, Hanau, Germany) connected to a Isoprime 100 isotoperatio mass spectrometer (Isoprime, Menchester, UK) - for elemental analysis2.3 ProcedureMaintenance and testsChecking of the background values, vacuum to prevent colliding, leakage by blowing pureArgon at equipment connection, stability, precision and performance of IRMS with referencegas pulses, linearity test with reference gas pulses and standards were done by supervisor.

Sample preparationDifferent sugar samples were grinded into small powder with pestle and mortar. Powder samples and standards were weighed in tin capsule, which were filled outside the balance. The weight and name of each sample and standard is recorded i a grid . Both blank and acetanilide are set between every 8 measurements, which are used to check whether the combustion and measurement are complete

3. RESULTSThe inhouse standard (Acetanilide) and samples were stored in a 96 well plate to identify thesamples inside folded capsules. The arrangement of the storage of sample and standardwith their respective weight is given in Table 1.Table 1. Storage of samples and standards in 96 well plate line

Two-point CalibrationThe actual value 13C values for USGS 40 (L-glutamic acid), USGS 41 (L-glutamic acid enriched in 13C) and Acetanilide were taken from website and literature.[ http://www.ciaaw.org/carbon-references.htm]. Their measured values with EA-IRMS with standard deviation and their actual value are shown inTable 2. Table 2. Actual and measured 13C values of USGS 40, USGS 41 and Acetanilide

The calibration curve is obtained by plotting actual 13C value vs measured value of USGS 40 and USGS 41 which is shown in Figure 2.

Figure 2. Two point calibration curve of USGS 40 and USGS 41

The actual values of sugars samples can be obtained using a equation of y=1.01172x - 36.882 obtained from excel of the calibration curve. The measured value with standard deviation and actual (calculated) 13C of sugar samples are shown in Table 3.

Sample Name13C+SD (Measured)(n = 3)13C ( )(Calculated

Sample 116.69989 0.03

-19.3339

Sample 226.94968 0.03

-9.20265

Sample 325.08659 0.02

-11.0442

Sample 425.03087 0.06

-11.0993

Sample 511.30589 0.05

-24.6655

Sample 69.449571 0.06

-26.5004

Sample 725.34254 0.07

-10.7912

Sample 810.29238 0.08

-25.6673

The 13C values of all samples according to its plant origin are shown in Fig 3

C4 PlantsC3 plantsCAM Plants

Figure 3: The 13C values of all samples according to its plant origin

4. Discussion

The calibration curve with USGS 40 and USGS 41 indicates that the internal standard used during EA-IRMS measurements closely fit in the two point linear calibration curve ensuring its consistency with two-point calibration curve. Hence acetanilide is applicable to use as internal standard along with samples for sample 13C measurements. The actual 13C of all sugars samples were obtained using two-point linear calibration curve. The 13C value obtained from sugars can be used to differentiate between their plant origins. The 13C values is in the range -23 to -29 for C3 plants, -10 to -15 for C4 plants and the values lies in-between for CAM plants[2]. According to Figure 3 sugar samples from 5, 6 and 8 are of C3 plant origin; 2, 3, 4 and 7 are of from C4 plants origin and 1 is of CAM-plants origin. Hence, it's possible to discover the origin plant source of sugar by EA-IRMS method. This kind of authentication of sugars (food products) helps consumers to buy the sugars in response to their choice reflecting their economical and health status. it can be concluded that the identification of sugars can be done by analyzing the carbon isotope by EA-IRMS.

6. Questions 1. Why packed columns are used to separate the gases?The packed columns are used because it allows the multiple element analysis of isotope ratios for entire sample. The sample gases are separated due to different retention time. For example, in our experiment , both N2 and CO2 flow into the packed column after reduction of NOx. The column here is long enough to separate N2 as waste gas which is pumped out after then, because the retention time of N2 is shorter than of CO2.. After N2 only CO2 enters the IRMS for the further carbon and oxygen isotope analysis.

2. Which requirements according to precision are necessary for carbon isotope ratio measurements?

- Precision can be attained by practical limitation of instrumental noise and the fundamental limitation of counting statistics. (The carbon can be diluted to prevent the misbalance of the ratio.)

3.Describe the difference between non-transient and transient signals.

5. REFERENCES

1. Lab manual: stable isotope analysis for food authenticit control . 2. Book: Compound specific stable isotope analysis : Maik jochmann and Torsten C. Schmidt.

3. Dr. MAIK A. JOCHMANN, 2014. Lectures materials of Stable isotope analysis.Instrumental analytical chemsitry, University of Duisburg-Essen.

4. ZHANG, L., KUJAWINSKI, D. M., FEDERHERR, E., SCHMIDT, T. C. & JOCHMANN, M.A , 2012. Caffeine in your drink: natural or synthetic? Anal Chem, 84, 2805-10.

5. http://www.ciaaw.org/carbon-references.htm