Food Analysis Exam 2
Why are external standards commonly used for HPLC (unlike in GC, for which internal standards are more commonly used)?
HPLC uses larger sample volumes and autosampler or sample loading loop allow for uniform volumes injected
The most common detectors for GC are TCD, FID, ECD, FPD, and PID. Differentiate each of these with regard to the operating principles. Also, indicate below which detector(s) fits the description given. a. Least sensitive b. Most sensitive c. Least specific d. Greatest linear range e. Nondestructive to sample f. Commonly used for pesticides g. Commonly used for volatile sulfur compounds
a. TCD b. ECD c. TCD d. FID, PID e. TCD f. ECD g. FPD
For each set of two (or three) terms used in chromatography, give a brief explanation as indicated to distinguish between the terms. a. Adsorption vs. partition chromatography b. Normal-phase vs. reversed-phase chromatography c. Cation vs. anion exchangers d. internal standards vs. external standards e. TLC vs. column liquid chromatography
a. adsorption: solid stationary phase, liquid or gas mobile phase, solute interact with phases w/ van der waals, electrostatic, hydrogen bonding, hydrophobic interactions partition: liquid stationary phase, liquid or gas mobile phase, solute partitions between liquid and stationary phases according to partition coefficient b. normal: polar stationary phase, nonpolar mobile phase, polar compounds elute last reversed: nonpolar stationary phase, polar mobile phase, nonpolar compounds elute last c. cation exchanger: negative charge and bound cations anion exchanger: positive charge and bound anions d. internal standards: stds not present in sample and added in constant amount to sample and standard. On std curve, plot ratio of peak height or area of internal standard (y) vs concentration of standard compounds (x) external standards: stds are same as present in samples, each standard is prepared of different concentrations and inject separate from sample. On std. curve plot peak height or area of each standard vs concentration of standards e. TLC: solid stationary phase in thin layer on plate or sheet with liquid at the bottom of the chamber. sample is applied as a dot on stationary phase at bottom of plate and bands by chemical reaction or fluorescence is how separated solutes are identified. Column liquid: stationary phase is liquid or solid inside column, while liquid passing through the column is mobile phase. sample is applied to top of column and detectors identify the separated solutes
Describe the mechanism of adsorption, partition, ion exchange, and affinity chromatography. Give an application example for each.
adsorption: stationary phase retains analytes, mobile phase moves analyte forwards, separate based on polarity, polar compounds are adsorbed tightly on silica and move slower than nonpolar compounds, polar solvent has a greater elution power than nonpolar solvent. application is synthetic food colorants partition: stationary phase is a liquid, surface of silica is coated with a liquid so analyte molecules are absorbed into the liquid; coated support vs bonded support, normal phases (polar bonded silica & nonpolar compounds elute first) or reversed phases (nonpolar bonded silica & polar compounds elute first) for bonded supports. application is sugar levels in foods ion exchange: positive ions attract and bond negative ions. separate ionic from nonionic compounds, separate cationic from anionic, separate mixtures of similarly charges species. cation exchangers have negatively charged surface while anion exchangers have positively charged surface. strong exchangers, elution is done by increasing ionic strength. weak exchangers, elution is done by changing pH. application is water treatment or separating proteins affinity: based on interactions between 2 components that are ideally suited to each other both electrostatically and spatially. most specific chromatographic method. separation is based on biochemical interactions. application is purification of enzymes or antibodies or glycoproteins
For a particular assay, your plot of absorbance vs. concentration is not linear. Explain the possible reasons for this.
analyte concentration may be too great so its absorptivity is altered. radiation passing through sample may be polychromatic.
What is gradient elution from a column, and why is it often advantageous over isocratic elution?
grading elution refers to changing the mobile phase (increasing the ionic strength or pH during elution to enhance resolution and decrease analysis time. isocratic: elute column with 30% methanol for 20 min, while gradient: linearly increase methanol content in mobile phase from 20-60% in 20 min.
The absorbance of 3.5x10-5 M cyanidin solution in a 0.5-cm cuvette at 520 nm is A=0.34, please calculate its molar extinction coefficient?
molar extinction coefficient ε = A/bc = 0.34/(0.5*3.5x10-5) = 0.194 x 10^5
At 520 nm, if 60% of light transmit through a cuvette and 40% of light is absorbed by the solution in the cuvette, please calculate transmittance T% and absorbance A.
%T = 60% A = log(1/T) = 0.222
A column has this label on it: Waters, C-18, 2.5 (internal diameter) x100 mm, 3.5 µm. Is this a reversed phase or normal phase column? What is the particle size of the silica in the column? Based on the standard flow rate of 1 mL/min on a standard column of 4.6 internal diameters, please calculate the appropriate flow rate for this column.
1/4.6^2 = x/2.5^2 flow rate (x) = 2.5^2/4.6^2 = 0.295 mL/min
What are the functions of a guard or pre- column for a HPLC column? Why is it recommended to filter the samples through 0.45 µm-pore filtration units before they are injected on a HPLC system?
Both have the function to protect and extend the life of a HPLC column. Filtration units remove small particles that may clot HPLC columns. A guard column is a short and disposable column that is made of the same material as the HPLC column. It retains small particles and other "dirty" compounds that may be irreversibly absorbed on the HPLC column.
What criteria should be used to choose an appropriate wavelength at which to make absorbance measurements, and why is that choice so important?
Choose the wavelength at which the analyte demonstrates maximum absorbance, and where the absorbance does not change rapidly with changes in wavelength, if possible. This provides maximum sensitivity (i.e., absorbance change per unit change in analyte concentration) and greater adherence to Beer's law.
Chlorophyll A has a uv/vis spectrum like this. Can you determine its λmax? What wavelength would you use to determine chlorophyll A content on a spectrophotometer?
Choose λmax and 2-10 nm bandwidth around it
Describe the working mechanisms of electron impact, electrospray, and APCI ionization; there advantages and disadvantages in applications.
EI: analyte is vaporized into MS ion source. vapor is impacted by beam of electrons with enough energy to ionize the molecule; electrons also fragment a whole molecule into pieces. pros- mass spectrum is fragment-rich, each compound has unique set of fragments, libraries of max spectra can be searched for compound identification, can interface with GC to make GC-MS. cons- sample must be thermally volatile and stable, harsh ionization, organic compounds are smashed into pieces by electron beam before entering mass analyzer, molecular ion may be weak or absent making it difficult to know molecular weight ESI: charged liquid is sprayed into electric field of high voltage, droplets shrink quickly, charge density increases and causes analyted to be ionized, generates [M+H]+ or [M-H]- in positive or negative mode. pros- good for charged polar compounds, very low chemical background leads to excellent detection limits, can interface HPLC with MS, soft ionization, compounds stay intact and molecular ions are highest peak cons- no good for uncharged low polarity compounds, compounds may lose or gain multiple H+ and become doubly, triply, or multiply charged ions APCI: a corona discharge electrode is used to ionize the analyte, benefits and limitation similar to ESI; pro- good for uncharged or low polarity compounds, can interface HPLC w/ MS, generates [M+H]+ or [M-H]- in positive or negative mode
Why you must degas the solvents before they can be used as HPLC mobile phases? How to degas solvents?
Gas in solvents form air bubbles and are not compatible with the high pressure of HPLC. Degassing can be done using an on-line degasser, sonication, or vacuum.
When you try to measure absorbance on a spectrophotometer, you find the solution in the cuvette is not clear. There is some turbidity. Can you use this to reliably measure the absorbance for quantitation purpose? If you insist to use it, will the content will be overestimated or underestimated? Explain
It cannot be used for quantitation. The content will be overestimated because turbidity in the solution blocks the light and causes an erroneously high absorbance reading.
Compound "zeinol" can be measured at 205 nm on a spectrophotometer. Can you use methanol (UV cutoff 210 nm) as a solvent to dissolve sample? Can you use methanol as mobile phase for HPLC using a UV detector for this compound? Why?
No, a solvent absorb all UV light at and below its UV cutoff wavelength. A solvent can only be used if the detection wavelength is above its UV cutoff.
In a particular assay, the absorbance reading on the spectrophotometer for one sample is 2.033 and for another sample 0.032. Would you trust these values? Why or why not?
No, the 2.033 value is too high and the 0.032 value is too low to be ideal for good precision. Ideally use a range of ~0.1 to 0.9 absorbance units. With too high or too low of values, relative concentration uncertainty is too high.
Why is it common to use absorbance values rather than transmittance values when doing quantitative UV-Vis spectroscopy?
T and %T are not directly proportional to the concentration of the absorbing analyte in the sample solution. Under appropriate conditions, absorbance is directly proportional to the concentration.
In an assay, the absorbance reading on the spectrophotometer for one sample is 2.60 and for another one 0.03. Would you trust and use them for quantitation purpose? Why or why not?
The 2.60 is too high and the 0.03 is too low for reliable quantification. The ideal range is 0.1-0.9 (a more conservative range is 0.2-0.8 on an older instrument)
UPLC has better performance than the HPLC. What is the mechanism?
UPLC uses columns packed with silica of much smaller particle size. Smaller particle size provides high separation power but requires high pressure to pump the solvent through column.
(a) You measure the percent transmittance of a solution containing chromophore X at 400 nm in a 1-cm path length cuvette and find it to be 50%. What is the absorbance of this solution? (b) What is the molar absorptivity of chromophore X if the concentration of X in the solution measured in question 2(a) is 0.5 mM? (c) What is the concentration range of chromophore X that can be assayed if, when using a sample cell of path length 1, you are required to keep the absorbance between 0.2 and 0.8?
a) A = -log T = -log 0.5 = 0.301 b) A/bc = ε = 0.301/(1*5*10^-4) = 602 c) lowest: c = A/εb = 0.2/602*1 = 3.3*10^-4 highest: 0.8/(602*1) = 1.3*10^-3
A particular food coloring has a molar absorptivity of 3.8 × 10^3 cm−1 M−1 at 510 nm. (a) What will be the absorbance of a 2 × 10−4 M solution in a 1-cm cuvette at 510 nm? (b) What will be the percent transmittance of the solution in (a)?
a) Use Beer's Law → A = εbc = (3.8*10^3)(1)(2*10^4) = 0.76 b) A= -log T T = 10^-A = 10^-0.76 = 0.1737 %T = 17.4
A sample containing compounds A, B, and C is analyzed via LC using a column packed with a silica-based C18 bonded phase. A 1:5 solution of ethanol and H2O was used as the mobile phase. The following chromatogram was obtained. A peak at 2 min B peak at 4 min C peak at 14 min a. Is this normal- or reversed-phase chromatography? Explain your answer. b. Which compound is the most polar? c. How would you change the mobile phase so Compound C would elute sooner, without changing the relative positions of Compounds A and B? Explain why this would work. d. What could possibly happen if you maintained an isocratic elution mode at low solvent strength?
a) reverse phase because stationary phase is nonpolar and mobile phase is polar b) compound A is most polar c. after ~6 min, change to less polar solvent this would make compounds A and B elute as normal, but then would make the mobile phase to be more like the stationary phase, so compound C which is nonpolar would elute sooner d. compound C might not elute
You plan to use GC to achieve good chromatographic separation of Compounds A, B, and C in your food sample. You plan to use an internal standard to quantitate each compound. By answering the following questions, describe how using an internal standard works for this purpose. a. How do you choose the internal standard for your application? b. What do you do with the internal standard, relative to the standard solutions for Compounds A, B, and C and relative to the food sample? Be specific in your answer. c. What do you measure? d. If you were to prepare a standard curve, who would you plot? e. Why are internal standards commonly used for GC?
a. not a compound present in the samples to be tested, has the same characteristics as compounds to be quantitated, elutes at a different time from compounds of interest b. add a constant amount of internal standard to food sample and to solutions of mixed standard compounds. Inject constant volume of sample for each type c. measure peak area or height d. [peak height of internal standard versus peak height of standard A, B, and C] vs. Concentration of A, B, and C e. sample volumes injected for GC are very small, so it is difficult to always inject the exact same volume
What is the base peak on a mass spectrum? What is the molecular ion peak?
base peak: fragment that has the highest abundance or intensity molecular ion peak: peak that has the highest mass to charge ratio and represents the charged intact molecule
What are the differences between binary and quaternary pumps for a HPLC system? Give example of four types of HPLC detectors and list the advantages and disadvantages of each. What detectors can be used to detect simple sugars, vitamin C, caffeine, and carotenes?
binary pumps: generates a binary gradient with 2 solvents, mixes the solvents at high pressure end, can tolerate tiny amount of gas in solvents so degasser is optional, provides better pressure and flow rate stability, more expensive quaternary pump: generates a quaternary gradient with 4 solvents, mixes the solvents at low pressure end, cannot work if solvents are not degassed, need a degasser to go with it. UV/Vis: simple, reliable, inexpensive, compatible with gradient elution and non-destructive; but not as sensitive as fluorescence detection, samples of use: carotenoids, flavonoids fluorescence: highly sensitive, low background, highly selective; but, changes in fluorescence can occur with pH and viscosity. fluorescence can be quenched by ions. samples of use: vitamin E, drugs, aflatoxins, epicatechin refractive index: detects any compounds, but lacks sensitivity, impractical for gradient elution, and unstable base line. sample use: sugars and oligosaccharides electrochemical: highly sensitive and specific, but large amounts of salts have to be added in mobile phase for conductivity & a pump with self-washing has to be used to wash away precipitated salts on the piston. destructive detection samples of use: ascorbic acid, isoflavones
The molar mass of delphinidin is 303 g/mole. You are requested to measure its content in a grape juice. One milliliter of grape juices is mixed with 9 mL of water. Measured A=0.43 using a standard 1-cm cuvette. The molar extinction coefficient of delphinidin is 5.34 x 105. Please calculate the content of delphinidin in this grape juice in mmole/L or mg/L.
c = A/εb = 0.43/ (5.34 x 10^5 * 1) = 0.805 x 10^-6 mol/L 10 x c = 8.05 x 10^-3 mmol/L (10 is dilution factor) 8.05 x 10^-6 mol/L * 303 g/mol = 2439 x 10^-6 g/L = 2439 x 10^-3 mg/L = 2.439 mg/L
Describe a circumstance in food analysis where you would use standard addition for quantitation. Describe a circumstance in food analysis where you would use internal standard for quantitation. In lab-7, was pure caffeine used as an external standard, an internal standard, or standard addition?
circumstance requiring standard addition: quantitation of a target compound in a sample cannot be done due to interfering compounds (matrix effect). Interference cannot be excluded or compensated on a spectrophotometer. circumstance requiring internal standard: injection volume for a GC analysis is too small to be accurate lab-7 used pure caffeine as an external standard
You are doing GC with a packed column and notice that the baseline rises from the beginning to the end of each run. Explain a likely cause for this increase.
column bleeding is caused by degradation of organic stationary phase at high temp
Why must sugars and fatty acids be derivatized before GC analysis, while pesticides and aroma compounds need not be derivatized?
compounds that must be derivatized are those that are thermally unstable, too low in volatility, or yield poor chromatographic separation due to polarity
What are the major differences in how ionization occurs in the electrospray versus the APCI interface? What is ion suppression?
electrospray: charged liquid is sprayed into an electric field of high voltage (4000 v common); droplets shrink quickly due to solvent evaporation; charge density increases and causes analytes to be ionized APCI: configuration and mechanisms is similar to electrospray; the only difference is that a corona discharge electrode is added into the spray path to ionize the analytes. ion suppression: ionization of analytes are suppressed by solvents or other matrix in the sample
Describe the applications and differences of using external standard, internal standard, or standard addition for quantitative analysis
external standard: most common, used in HPLC & spectroscopy standard addition: overcomes matrix interference, used in spectroscopy internal standard: overcomes inaccurate injection, used in gas chromatography
What type of detectors can be used to selectively detect volatile compounds that contain sulfur, phosphorus, or halogens in GC analysis? What type of detector can be used to detect CO2?
flame photometric: selectively detect sulfur or phosphorous with excellent sensitivity and good linear range electron capture: selectively detect halogens, nitros with excellent sensitivity and narrow linear range flame ionization can detect CO2 with good sensitivity and excellent linear range
If a compound can be measured using either UV/vis spectroscopy or Fluorescent spectroscopy, what would you choose? What are the advantages of fluorescent spectroscopy over the UV/vis spectroscopy?
fluorescent spectroscopy because has a much lower LOD than UV/VIS and is more specific than UV/VIS
What is advantage of bonded supports, or bonded phase over coated supports for partition chromatography? What is the most common support in partition chromatography? What are the differences between reversed phase and normal phase chromatography in terms of the nature of stationary phase, mobile phase, and what elute first?
for bonded phase supports the stationary phase is covalently attached to the support material, so the stationary phase is not stripped off with continued use of the column. The most common support is silica. normal phase: polar stationary phase (silica), nonpolar mobile phase (hexane-ethyl acetate mixture), & nonpolar compounds elute first reversed phase: nonpolar stationery phase (C18), polar mobile phase (methanol-water), & polar compounds elute first
What is the primary function of an HPLC detector (regardless of type)? What factors would you consider in choosing an HPLC detector? Describe three different types of detectors and explain the principles of operation for each.
primary function: translates concentration changes in the HPLC column effluent into electrical signals factors to consider in selecting detector: solute type & concentration, sensitivity, linear range, selectivity, compatibility with solvent, effect of temp or flow rate changes, possibility of use with gradient elution, initial and operating costs UV-VIS absorption detector: measures absorption of radiation in UV and visible wavelength range according to Beer's law, selective Fluorescence detector: measures the emission of electromagnetic radiation by molecules that fluoresce, more sensitive and selective than UV detector Refractive index detector: measures change in refractive index of mobile phase due to presence of solutes, universal detector, sensitive to changes in temp and flow rates, cannot be used with gradient elution Amperometric detectors: measure the change in current as analyte is oxidized or reduced by the application of voltage across electrodes of the flow cell, sensitivity and selectivity improved by adding pulse techniques Conductivity detector: responds to presence of ions eluting from the column
Describe the working mechanisms of quadrupole, triple quadrupole, 3D ion trap ion analyzer, and Orbitrap.
quadrupole: quadrupole is an ion filter, an ion with certain m/z is allowed to pass through at a given voltage. when the voltage is ramped low to high, ions with m/z low to high fly through and are detected. (mass scan) by selecting 1 or more voltages, 1 or more ions are allowed to fly through and be detected (selected ion monitoring) triple quadrupole: mass filter allows a specific ion or range of ions to pass, collision cell filled with nitrogen. analytes collide with N2 gas and breaks into pieces (fragmentation). Mass filter, allows a range of ions or a specific ion to pass 3D ion trap: set the ion trap to ion trap of a range m/z, ramp EM field to allow ions to fly out, keep an ion of interest in the trap, give energy pulse to ion and break ion into pieces, ramp EM field to allow the ions to fly out Orbitrap: has an outer barrel-like electrode and inner spindle-like electrode, ion from C-trap is injected and trapped around the spindle in an orbital motion, mass spectrum is obtained by Fourier transform of the frequency signal of spinning ions; superior mass accuracy, superior resolution, excellent dynamic range and sensitivity
What are the basic components of a MS?
sample introduction → ion source → mass analyzer → detector → data system
In fluorescence spectroscopy, why is the wavelength of the emitted radiation longer than the wavelength of the radiation used for excitation of the analyte?
the wavelength of the emitted light is longer because typically some of the energy associated with the absorbed light is dissipated as heat (vibrational relaxation) and, thus the emitted light is of lower energy. The lower energy associated with the photons of the emitted light means that the corresponding light waves are of longer wavelength.
Why is the injection port of a GC at a higher temperature than the oven temperature?
to rapidly vaporize the sample so the sample can pass through the column for separation
What are criteria of choosing an internal standard? What is the advantage of using internal standard? Give an example about how to use internal standard for quantitation.
use of internal standard minimizes errors due to sample preparation, apparatus, and operator techniques. Injection volume does not need to be accurately measured
How can chromatographic data be used to quantify sample components?
use peak height, peak area, or peak mass to plot against concentration
