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
