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49 Cards in this Set
- Front
- Back
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HPLC is most important for 2 general things. State both. |
Isolating and purifying biomolecules |
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The sample will interact differently to 2 things based on it's affinity to each, state the name of both. |
Mobile phase and stationary phase |
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What does HPLC stand for? |
High Performance Liquid Chromatography |
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State 4 biomolecules that HPLC can separate? |
Answer can include any 4 of the following: Amino acids, carbohydrates, lipids, nucleic acids, proteins and peptides |
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For instrumentation, how many ml must the resirvoir hold? What must happen to the solvent before entering? |
500mL, degassed and filtered |
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State 3 solvents can be used. |
MeOH, CH3CN or H2O |
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What 2 solvent systems can be used? |
Isocratic (1 solvent used) or Gradient system (mix of 2 solvents that changes) |
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At what pressure range (psi) does the pump operate? What flow rate is seen for an analytical pump and a preparatory pump? |
500-5,000 psi Analytical: 1mL/min Preparatory: 100mL/min |
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The injector involves a microsyringe being injected into what? |
Neoprene/teflon septum |
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What is the typical volume of an analytical injector and a preparative injector? |
Analytical: 1 microL to 5 mL Preparative: 100 microL to 20 mL |
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What 3 sizes are available for the diameter of analytical columns? |
2.1, 3.2 and 4.5 mm |
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Up to what diameter are preprative columns seen? What range of lengths are seen for columns? What is used to prevent impurities from entering the column? |
30mm 10-20cm A Guard column |
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What 2 form of detectors are used? Which one is more sensitive? Which one produces a linear response? (What can a linear response tell us?) |
Photometric: linear response-allows quantity to be given Fluorescence: more sensitive |
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For the collector, what is used to allow the sample to run directly into the ESMS? |
Splitter |
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What is the run time of the HPLC? |
1min to 30mins |
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For a reverse phase C18 coulmn, what will exit the coulmn first? What will exit last? |
Exit first: Most hydrophillic Exit last: Most hydrophobic |
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In the results from the HPLC, what does the follwoing indicate: to tR K' |
to: point that sample is loaded tR: time 1st peak has come off K': Tells where peak 1 will be in relation to the void volume |
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How is K' calculated? |
K'=(tR-to)/to |
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Resolution is Rs. What does this mean and how is it calculated? What is an ideal value for Rs? |
Measure of how well 2 peaks have seperated Rs=2(tR2-tR1)/(tW1+tW2) Ideal: 1.5 |
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T stands for tailing factor. What does f have to do with this here? What would be an ideal relationship between tW and f? |
f is width from mid linr to start/end of peak Ideally tW=2f (Gaussian distribution) |
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What does N stand for? How is this calculated? What is it a measure of? |
N: Theoretical plate number N=16(tR/tW)^2 Measure of column efficiency |
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To find a quantitative measure of how much of each compound is present, 3 methods can be used. The first is integrating Gaussian distribution and calculating the area underneath, is narrow, just use height. What are the other 2 methods? |
Produce a standard curve Inject internal standard |
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What is the main difference between preparative and analytical HPLC? |
Preparative: a purification technique Analytical: Identify components and quantify |
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The statonary phase can be porous beads or microporous beads. For porous beads, what comprises their thin porous outer shell? What is their diameter? |
Silica, alumina, ion exchange resin or chemically bonded plates 20-45 microm |
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For microporous beads, long pores or short pores are seen. What are the diameter of both types of beads? Which is most common? |
Long: 20-40 microm Short: 5-10 microm (most common) |
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For the normal phase, what type of molecules get retained, what type go through on the mobile phase? |
Retained: Hydrophillic/highly polar molecules Pass through: hydrophobic/low polarity |
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For normal phase, state 4 solvents that can be used? |
Answer can include any 4 of the follwoing: Hexane, EtOAc, MeOH, MeCN, CHCl2 |
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For normal phase, state 4 things that can be separated? |
Answer can include any 4 of the following: Steroids, Alcohols, Organic Acids, Vitamins and Pesticides |
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For reverese phase, what type of molecules get retained and what pass through quickly? |
Retained: Hydrophobic/Low polarity Pass through: Hydrophilic/High polarity |
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State 4 things that can be seperated using reverse phase HPLC? |
Answer can include any 4 of the following: Peptides, nucleotides, carbohydrates, amino acids and derivatives |
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Ion Exchange HPLC seperates based on what? in an anionic column, which type of molecule will be retained and which will pass out? |
Based on charge, Retained: -ve charge will make an ionic bond with Nitrogen+ atom Pass through: +ve charge or neutral |
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In a cation Ion Exchange column, which type of molecule will be retained and which will pass out? |
Retained: +ve charge will make ionic bond with Sulfates- Pass through: -ve or neutral |
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How are the retained molecules unstuck? What is an Ion Exchange column used to separate? |
Disrupt with salt Separate proteins and peptides |
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Gel exclusion aka size exclusion seperates proteins and nucleic acids up to what size? (Da) What size difference is needed for effective separation? |
Up to what size: 10,000,000 Da Separates between: 5,000 Da |
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A chiral HPLC separates enantiomers. What is modified to achieve this? |
Add protein to surface of bead, 1 enantiomer will interact differently in chiral environments |
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What is the definition of derivitisation? |
Increasing the detection limit of samples or improving separation |
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To improve derivatization and increase sensitivity, the separation process can be altered. Give an example of how this can be achieved. What other 2 things can be done? |
Seperate enantiomer using RP-HPLC and FLEC, the diastereoisomer product can seperate using a normal column Add fluorescent group, increase UV absorbtion |
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In gas chromatography, what is in the mobile phase? What is in the stationary phase? |
Mobile: gas Stationary: liquid |
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How is derivitisation acheived in gas chromatography? Give 2 examples. |
Addition of groups that make the molecule more volatile. Examples can include any 2 of the following: Silylation of acids: produce ethers and esters Acetylation of amines: produce CF3C=O Alkylation of fatty acids: produce methyl, ethyl and butyl |
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In the instrumentation of GC, what is the gas supply? What conditions have to be met with this gas? |
N2, Ar or He Pure, dry and chemically inert |
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In the sample injection port, what must occur? What volume does this port hold? |
Sample muct be volatised 0.1to 20 microL |
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What is the coil comprised of? What range of diameters can it be? How long can it be? |
Fused silica capillary 0.1mm to 0.53mm wide Up to 20m long |
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What does WCOT stand for? |
Wall Coated Open Tubular |
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what occurs in the detector of GC? |
Flame ionisation, ions produced and detected by pair of polarised electrodes |
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State 2 advantages of using HPLC over GC. |
-Wide range of compounds can be detected, including thermally liable, highly polar, high MW and proteins -Compounds not destroyed |
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State 2 disadvantages of using HPLC over GC. |
-Cost of solvents -Optimisation of column, solvent and flow rate is tricky |
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State 2 advantages of using GC over HPLC. |
Very sensitive Little compound needed |
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State 2 disadvantages of using GC over HPLC. |
Compounds must be thermally labile Compounds destroyed |
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What are the main uses of: HPLC GC? |
HPLC: Detection of chemicals and sports medicine GC: Purification and analytical |
