exp 4 post lab Flashcards
Thin Layer Chromatography
- the higher the affinity of the compound with the mobile phase, the ______ it will move
- the lower the affinity of the compound with the mobile phase, the ____ it will move
- faster and higher
- slower and lower
TLC
- 3 main steps
- Preparation of TLC plate
- Preparation of Plate Spotters and Spotting the Plates
- Developing a TLC plate
TLC - Preparation of TLC plate
- stationary phase used is:
- ^^ preferred because its?
- 2 types of ^^ + definition + when used
- preparation of ^^ involves the use of _____ and _____ to prevent _____
- silica gel (made of silicon dioxide joined together)
- higher sample capacity
- Hydrophilic silica
- used when mobile is nonpolar
- polar surface - Hydrophobic silica
- used when mobile is more polar than stationary phase
- nonpolar surface
- chemically modified
- face mask and gloves to prevent silicosis
TLC - Preparation of Plate Spotters
4 types of spotters used?
- Capillary Tubes
- Stirring Rod (not recommended)
- Toothpick
- Inert pointed materials
TLC - Spotting the Plates
- the sample is spotted ____ cm to ____ cm from the _____ _____ _____
- done by _____ pressing the _____ or using a ____
- 1 cm to 2 cm
- plate’s bottom edge
- manually
- spotter
- syringe
TLC - Developing a TLC Plate
- 3 steps + importance (1; 1; 3)
- Placing of filter paper inside the developing chamber/beaker and equilibration
- ensures that the container is saturated with solvent vapors evenly (for effective separation)
- Pouring solvent below the sample spots
- prevents dissolution of sample
- Addition of 2 drops glacial (pure) acetic acid to the developing chamber containing the mobile phase
- prevents “tailing” (changing directions of spots)
- pampabigat ng solvent
- maintains low pH (silica gel plate is acidic) and prevent reactions that may form new products/compounds = error in separation
Pano kung masyadong mababa sa 1 - 2 cm yung pag spot sa TLC silica gel plate?
pwedeng ma dissolve yung pigments by the mobile phase rather than tumaas
Sample used in TLC
Malunggay leaves
Relevance of POUNDING malunggay leaves using mortar and pestle + what was added
+ why was this added??
ETHYL ACETATE
- extracting solvent
- very volatile
- break cells of the leaves and release the pigments into the ethyl acetate
pigments are soluble in ethyl acetate
2 possible mobile phases used in TLC
- what is the basis for choosing the better solvent (3)?
- what was chosen at the end?
- 8:2 petroleum ether: ethyl acetate
- 1:1 petroleum ether: ethyl acetate
v/v
- separated the highest number of components
- least degree of overlaps
- clearest color of the spots
8:2 was the best solvent
MOBILE PHASES IN TLC
- usually a combination of ____ and _____ compounds
- if the stationary phase is POLAR, the ______ compound in the mobile phase will have larger proportions
- if the stationary phase is NONPOLAR, the ______ compound in the mobile phase will have larger proportions
in the experiment, which is polar and nonpolar in the mobile phase used?
- polar and nonpolar
- nonpolar
- polar
polar (ethyl acetate)
nonpolar (petroleum ether)
TYPE OF CHROMATOGRAPHY USED IN TLC
+ stationary and mobile phase used
NORMAL PHASE
- stationary is polar (hydrophilic silica)
- mobile is nonpolar (8:2 petroleum ether:ethyl acetate)
ORDER OF SEPARATION OF PIGMENTS in TLC (2)
in normal & reverse phase?
NORMAL PHASE
- less polar components: carried farther from the origin (at the top)
- more polar components: separate at earlier times (at the bottom)
REVERSE PHASE
- more polar components: carried farther from the origin (at the top)
- less polar components: separate at earlier times (at the bottom)
PIGMENTS separated from MALUNGGAY LEAVES (5)
+ order of increasing polarity
+ order of separation (lower to higher spots) - normal
+ order of separation (lower to higher spots) - reverse
- Chlorophyll a
- Chlorophyll b
- Carotenoids
- Xanthophylls
- Pheophytins
INCREASING POLARITY:
Carotenoids < Xanthophylls < Pheophytins < Chlorophyll a < Chlorophyll b
NORMAL - ORDER OF SEPARATION L->H:
Chlorophyll b > Chlorophyll a > Pheophytins > Xanthophylls > Carotenoids
REVERSE - ORDER OF SEPARATION L->H:
Carotenoids > Xanthophylls > Pheophytins > Chlorophyll a > Chlorophyll b
MALUNGGAY LEAVES - PIGMENT
Chlorophyll
- ____ pigments present in ___
- 2 types + color + polarity
- major
- plants
- Chlorophyll a
- green and more intense color
- less polar - Chlorophyll b
- yellow-green
- more polar
MALUNGGAY LEAVES - PIGMENT
Carotenoids
- polarity
- color
- nonpolar
- yellow-orange
MALUNGGAY LEAVES - PIGMENT
Xanthophylls
- _____ (color) pigments produced when _____ are ____
- yellow
- carotenes
- oxidized
MALUNGGAY LEAVES - PIGMENT
Pheophytins
- ____ derivatives that lack ____
- polarity
- color
- chlorophyll
- magnesium
- nonpolar than chlorophylls
- gray
PC (Paper Chromatography)
6 samples used?
- Blue
- Red
- Green
- Yellow
- Violet
- Orange
PC - STATIONARY PHASE
- stationary phase used?
- where is ^^ adsorbed
WATER is stationary phase
- adsorbed on Whatman No.1 Filter Paper
PC - WHATMAN NO.1 FILTER PAPER
- a grade ___ quality
- the most _____ ____ paper due to ____ ___ and ____ rate
- composition?
- contains how much water by mass?
- 1
- used filter paper
- medium retention
- flow rate
- specially selected cotton cellulose
- 23% water
when water is adsorbed to cellulose (whatman no.1 filter paper), it results in a ___-____ ____ that is bonded with ____ ____
water-cellulose complex
- hydrogen bonding
PC - MOBILE PHASE
- what is the mobile phase used in the experiment?
- what other mobile phase can be used? + is this more polar than water like the salt?
- 2% NaCl
- isopropyl alcohol can be used: less polar than water
*lahat ng mobile phase maliban sa SALT ay LESS polar than water
which is more polar in PAPER CHROMATOGRAPHY: mobile or stationary?
THUS, what type of chromatography was done in PC?
MOBILE PHASE (2% NaCl) is more polar than STATIONARY (water)
so REVERSE CHROMATOGRAPHY was used
NaCl (mobile phase) has permanent ____ charges
Water (stationary) has permanent ____ charges
this explains that?
+ what bonds is prese
NaCl - full
Water - partial
thus, NaCl is more polar than water
PC - REVERSE CHROMATOGRAPHY
- again, why is paper chromatography reverse chromatography?
- order of separation (what is carried farther?)??
- mobile phase is more polar than stationary phase
ORDER OF SEPARATION
- the more polar components of the food dyes will be carried farther from the origin
PC - PIGMENTS SEPARATED
- 3 types of components that should emerge from all the food dyes?
+ color it exhbitis
+ presence or absence of polar groups
+ number of charged groups and relationship with mobile phase
- BRILLIANT BLUE FCF
- blue
- presence of polar groups
- has the most charged (7) groups associates the STRONGEST with NaCl mobile phase - TARTRAZINE
- yellow
- presence of 2 polar groups
- second most charged groups (6) : associates the second strongest with NaCl - ALLURA RED AC
- red
- presence of 1 polar group
- fewest charged groups (4)
PC
The negative charges of the food dye components will interact with the ____ of the NaCl
The positive charges of the food dye components will interact with the ____ of the NaCl
both of which will make _____ bond
- positive Na
- negative Cl
ion-dipole bonds
PAPER CHROMATOGRAPHY - RESULTS
- order of increasing polarity
- order of separation (lowest to highest)
- order of separation in reversed chroma
- order of separation in normal chroma
- allura red ac < tartrazine < brilliant blue fcf
- allura red ac > tartrazine > brilliant blue fcf
- allura red ac > tartrazine > brilliant blue fcf
- brilliant blue fcf > tartrazine > allura red ac
Paper of Chromatography
BASIS OF SEPARATION
ionic character
more ionic groups = more polar
IONIC CHARACTER
- found in what type of chromatography
- ____ in a compound as the number of ____ ____ increases
- paper chromatography
- increases
- charged groups
NOTES to remember what groups are more polar and less polar
MOST POLAR
- charged groups (O- or N+)
- COOH (carboxylic acid)
- CONH2 (amide)
- OH
- NH2
- CONHR
- NHR
- CONR2
- COOR
- CO
- ROR
- NR2
LEAST POLAR
OH > NH > ON > O > N
2 possible ERRORS in chromatography
+ reason (2 per error)
+ how to counter and why
- Use of ballpen for sample spots
- ballpen ink will smear with spot
- ballpen ink also consist of colored pigments
INSTEAD, use pencils since pure graphite lang siya which are made of inert carbon
- Level of solvent is above the sample spots
- no separation will occur
- solvent will wash away/dissolve the pigments
INSTEAD, make sure the solvent will not exceed sample spot
for a successful and effective chromatographic separation, ___ ___ of ____ ___ among the ___ participants in the separation process, which are the??
- proper balance of IMFA
- 3 participants
- solute/compound to be separated
- mobile phase
- stationary phase
selection of an appropriate solvent system/mobile phase is by ____ and ____
trial and error
How many cm / mm?
1 cm = 10 mm
If whatman no.1 filter paper ang gamit sa paper chromatography, automatic what is the stationary phase?
water
