New tools in food analysis:Spectrometry and mass

spectrometry;Capillary electrophoresis.

ARID AGRICULTURE UNIVERSITY

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Food analysis:

•Food analysis is the discipline dealing with the development, application and study of analytical procedures for characterizing the properties of foods and their constituents.

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New tools in food analysis:

• Spectrometry

• Mass spectrometry

• Capillary electrophorasis

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Spectrometry

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• Ion-mobility spectrometry• It is an analytical technique used to separate and

identify ionized molecules in the gas phase based on their ion mobility in a carrier buffer gas

• Mass spectrometry• Is an analytical technique that measures the mass-

to-charge ratio of charged particles• Optical spectrometry • Is a technique for measuring the distribution of light across the

optical spectrum, from the UV spectral region to the visible and infrared. 5

Spectrometry

Spectrometer:

• A spectrometer is an apparatus to measure a spectrum.

• Generally, a spectrum is a graph that shows intensity as a function of wavelength, of frequency, of energy, of momentum, or of mass.

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Types of spectrometers:

1. Optical spectrometer: (often simply called "spectrometers"), in particular,

show the intensity of light as a function of wavelength or of frequency.

1. Magnetic spectrometers:

2. spectrophotometer 

• It Is a spectrometer that only measures the intensity of electromagnetic radiation (light)

• Example:

•  mass spectrometers.7

Spectrometer use in food analysis:

• Mass spectrometer :• for characterization and estimation of food

components

• Gas chromatography mass spectrometer:• analysis of natural volatiles and reprivatized polar

isolates• chemistry of flavor and aroma• Isotop mass spectrometer :

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Isotop mass spectrometer :

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Mass spectrometry

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Mass spectrometry:

• (MS) is an analytical chemistry technique that helps identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions.

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A mass spectrum

• is a plot of the ion signal as a function of the mass-to-charge

ratio.

• The spectra are used to determine the elemental or isotopic

signature of a sample,

• the masses of particles and of molecules,

• and to elucidate the chemical structures of molecules, such

as peptides and other chemical compounds.

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Design of mass spectrometer

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Mode of action:

A sample, solid, liquid, or gas, is ionized, by bombarding it with

electrons.

cause some of the sample's molecules to break into charged

fragments.

These ions are then separated according to their mass-to-charge ratio,

by accelerating them and subjecting them to an electric or magnetic

field: ions of the same mass-to-charge ratio will undergo the same

amount of deflection.

The ions are detected by a an electron multiplier.

Results are displayed as spectra of the relative abundance of detected

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Applications:

• MS has both qualitative and quantitative uses.

• Identifying unknown compounds,

• Determining the isotopic composition of elements in a

molecule,

• Determining the structure of a compound by observing its

fragmentation.

• Other uses include quantifying the amount of a compound in a

sample or studying the fundamentals of gas phase ion

chemistry (the chemistry of ions and neutrals in a vacuum).15

Examples of application

1. Protein characterization:The two primary methods for ionization of whole

proteins are• electrospray ionization (ESI) and• matrix-assisted laser desorption/ionization (MALDI).

• Two approaches are used for characterizing proteins

a) "top-down" strategy of protein analysis.

b) peptide mass fingerprinting (PMF)16

Continue..

• "top-down" strategy:• intact proteins are ionized by either of the two

techniques described above, and then introduced to a mass analyzer.

• peptide mass fingerprinting (PMF),• proteins are enzymatically digested into smaller

peptides using trypsin or pepsin, either in solution or in gel after electrophoretic separation. The collection of peptide products are then introduced to the mass analyzer.

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Isotope ratio MS: isotope dating and tracing:

• Mass spectrometry is also used to determine the isotopic composition of elements within a sample.

• isotope ratio mass spectrometers (IR-MS) are used.

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Glycan analysis:

• (MS), has been predominantly used for characterization and elucidation of glycan structures

• Intact glycan may be detected directly as singly charged ions by matrix (MALDI-MS)

• Electrospray ionization mass spectrometry is used for the smaller glycan.

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Other examples

• Atom probe:

• Pharmacokinetics:

• Space exploration:

• Respired gas monitor:

• Trace gas analysis

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Pros and cons..

• Advantages of MS:• Increased sensitivity over

because the analyzer,

• Excellent specificity

• Information about molecular weight isotopic abundance of elements.

• Temporally resolved chemical data.

• Disadvantages of MS:• fails to distinguish between

optical and geometrical isomers and the positions of substituent in o-, m- and p- positions in an aromatic ring.

• Also, its scope is limited in identifying hydrocarbons that produce similar fragmented ions.

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Capillary Capillary ElectrophoresisElectrophoresis

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Introduction • Capillary electrophoresis (CE) is a relatively new, powerful

separation technique that is ideally suited for handling small amounts of sample material.

• Used in bioanalytical research, e.g., in biotechnology and in various clinical, diagnostic, genetic, and forensic applications

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WHAT IS CAPILLARY ELECTROPHORESIS

• In practical terms, a positive (anode) and negative (cathode) electrode are placed in a solution containing ions.

• Then, when a voltage is applied across the electrodes, solute ions of different charge, i.e., anions (negative) and cations (positive), will move through the solution towards the electrode of opposite charge.

• Capillary electrophoresis, then, is the technique of performing electrophoresis in buffer-filled, narrow-bore

capillaries, normally from 25 to 100 pm in internal diameter (ID).

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Continue..

• Separation by capillary electrophoresis can be detected by several detection devices. The majority of commercial systems use UV or UV-Vis absorbance

• a section of the capillary itself is used as the detection cell.

• The use of on-tube detection enables detection of separated analytes with no loss of resolution.

• detection, however, must be optically transparent

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• In general, capillaries are coated with a polymer (frequently polyimide or Teflon) for increased flexibility.

• The portion of the capillary used for UV detection, however, must be optically transparent

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Capillary Electrophoresis – mode Capillary Electrophoresis – mode of actionof action

• Electrophoresis in a buffer filled, narrow-bore capillaries

• Each capillary is about 25-100 μm in internal diameter

• When a voltage is applied to the solution, the molecules move through the solution towards the electrode of opposite charge

• Depending on the charge, the molecules move through at different speeds– Separation is achieved

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cont.cont.

• A photocathode is then used to measure the absorbencies of the molecules as they pass through the solution

• The absorbencies are analyzed by a computer and they are represented graphically

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Cont….

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Capillary Electrophoresis ApparatusCapillary Electrophoresis Apparatus

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CONT>>>>

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Similarities with HPLCSimilarities with HPLC

• CE offers several similarities to high performance liquid

chromatography (HPLC),

• ease of use,

• high resolution,

• speed,

• on-line detection,

• and full automation capability.

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CE and HPLC

• Analogous to HPLC, CE is also often referred to as

• high performance capillary electrophoresis

(HPCE). • the power supply is equivalent to an HPLC

pump and the capillary is equivalent to a column, the instrumentation is completely analogous.

• software packages used to control most commercial CE instruments are based heavily on existing HPLC software. 34

The Electropherogram

• The data output from CE is presented in the form of an electropherogram, which is analogous to a chromatogram.

• An electropherogram is a plot of migration time vs. detector response.

• The detector response is usually concentration dependent, such as UV-visible absorbance or fluorescence.

• The appearance of a typical electropherogram is shown in Figure for the separation of a three component mixture of cationic, neutral and anionic solutes.

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Applications

• Analysis of carbohydrates

• Analysis of inorganic anions/metal ions

• DNA profiling

• Protein identification

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Analysis of carbohydrates• Analysis of underivatized carbohydrates (arabinose, ribose,

xylose, inositol, galactose, glucose, fructose, and mannitol) is achieved using a CE-ESI-MS method based on the use of a highly alkaline running buffer for CE separation, and MS detection in the negative ion mode. LODs were in the range of 0.5–30 mg/mL.

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Advantages and disadvantages

• Advantages:• There is no mass transfer

between mobile and stationary phases

• Altering column conditions allows focusing or concentration of samples.

• Fast• Small Sample• Relatively inexpensive• Automated

• Disadvantages:

• Cannot identify neutral species

• Joule Heating• Cannot discern shape

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Summary

1. CE is based on the principles of electrophoresis. 2. The speed of movement or migration of solutes in CE is

determined by their 3. size and charge. Small, highly charged solutes will migrate

more quickly than large, less charged solutes.4. The data output from CE is called an electropherogram.

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Conclusion

• It is the most efficient separation technique available for the analysis of both large and small molecules.

• DNA Profiling, protein identification,

inorganic metals and ions can be detected easily by this method.

•

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References

• http://www.chemistry.or.jp/gakujutu/bcsj/bc-cont/b98nov_gif/kea1009con.gif

•  www.http.wikipedia.com• "Mass Spectrometry: A Foundation Course", K. Downard,

Royal Society of Chemistry, UK, 2004.• "An Introduction to Biological Mass Spectrometry", C. Dass,

Wiley, USA, 2002.• "The Expanding Role of Mass Spectrometry in

Biotechnology", G. Siuzdak, MCC Press, San Diego, 2004.

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