EFFECTS OF TIIE POLARITY 0F THE MOBILE PHASE IN PAPER CHROMATOGRAPIIY

John Panizza
9/19/2019

INTRODUCTION: The goal of the experiment was to show that paper chromatography can be
used as an analytical separation technique to detemine the specific number and types of
chemicals present in a dye mixture from non-toxic markers. The purpose of the lab was to
illustrate the effects of solvent polarity on dye solubility and separation patterns by completing
two chromatography runs, each using a different mobile phase of varying polarity. Two different
color dyes were analyzed in both chromatography runs, pen 1 (brown dye), and pen 2 (red dye).
Cellulose chromatography paper was used as the polar stationary phase, and the solvents used for
the mobile phases were distilled water and 5% sodiuni chloride (Nacl) solution.
Paper chromatography is useful because it is a relatively quick process and requires only
small quantities of material. Through capillary action, the mobile phase travels up the stationary
phase and the dye molecules that are less polar start to stick to the paper, whereas the dye
molecules that are more polar will stay soluble in the mobile phase until they adhere further up
the chromatography paper. The distances of the solvent front line and each individual pigment
spot from the solvent start line are used in a formula to calculate the retention factor (Rf). The Rf
is used to identify the individual chemicals present in the dye mixture. A low Rf value means
that the spot (dye molecule) stayed close to the initial starting point of the spot throughout the
chromatography run, which generally means the compound is not very polar or possibly
nonpolar (depending how low it is on the chromatogram), and it's strongly attracted to the polar
stationary phase. A high Rf value indicates that the spot traveled very far up near the solvent
front line. Additionally, high Rf values indicate the compound is very polar, has relatively weak
attraction to the stationary phase, strong attraction to the mobile phase, thus good solubility in
the mobile phase. Depending on the polarity of the dye being analyzed, increasing the polarity
of the solvent used in the mobile phase may allow for improved separation of the dye, therefore
allowing more accurate identification and total count of the dye mixture' s constituent chemicals.

PROCEDURE: The procedure followed for this experiment was exactly how it was written in
the lab handout. Please refer to pages 33-34 from the URI CHM 102 Fall 2019 Laboratory
Manual.
 Experiment 2: Procedures and Data Sheet
(AIlowed on concept review, pass in with your concept review & results table)
© Name: Date: 1`11lllsection.

TA Signature:

All data must be written in pen at the time it is collected. Pencil is not allowed!!
Record all measurements with the correct number of significant figures and units.
TA signature & TA initials on any changes made to the data are required or the data is invalid.

Part 1: Determination of Rf values for the nontoxic inks used in markers

Start this part of the lab before you do the concept review.
Use Figure 1 in the introduction to see the correct placement of lines and marking spots.

1. You will need 2 strips of chromatography paper approximately llcm long. Prepare each
strip by drawing a line on the paper 1.0 cm from the bottom with a pe±±£jJ. Draw a second
line lcm from the top as well. (Do not use a pen because the ink may be soluble in water
and interfere with the experiment by also separating and moving up the paper.)

P.tFh°:dsjtdheef::Pnegr}eonu:thcwajrseefut,1;nm:R:°|ddtohtefrpoafeer#'eany:tnf::tewp'::ctj[ijnpee::j'eTcahrt,::

I ofthefold in thechromatographypaper. The dotshould beapproximatelythisfize: (.)

3. Record the color of each pen in pencil below each dot. Also record the colors on this
sheet.

Penl:Color . 8p~

Pen2: Color . fuck

4. Fill a wash bottle with distilled water. Dispense 5 mL of distilled water into a 10 mL
graduated cylinder. Pour the water into a loo or 150 mL beaker.

5. Lower one of the spotted chromatography papers into the beaker until the bottom of the
paper is in the water. When you insert the paper into the beaker, THE DOT OF DYE MUST
BE ABOVETHE LEVEL OF THE LIQUID. Do not allow the spotted sample to touch the liquid
or the sample may dissolve ruining your experiment. Make sure the paper stands upright
on its own. Your paper may lean towards the side of the beaker, but the damp edge of
the chromatogram should not come in contact with the beaker at any time during the
separation.

6. Using a second beaker, repeat the process with 5 mL of the 59/o NacI solution.

7. The solvents will begin moving up the paper by capillary action. When the mobile phase
reaches the 1 cm line at the top of the paper, remove the chromatogram from the beaker
and lay it on a paper towel. Blot gently with another paper towel to stop the movement
of the liquid through the paper. It takes approximately y2 hour for each chromatogram
to develop.

8. The solvent Front (see introduction) may not be a perfectly straight line or may be slightly

® above or below the top lcm line that you drew. Before the chromatography paper has
completely dried, mark the Solvent Front with a second pencil line.

33
 9. Circle the colored spots in pencil immediately, as they often fade as they dry. Ideally the
spot w"I be completely spherical, but often it is more of a streak. Make your circle around
the darkest area of the spot as this is where most of the dye is located. (Fig 2)

10. Measure the distance traveled by the solvent from the bottom pencil line to the solvent
front. This measurement will be recorded as "Distance traveled by the solvent" for use
in the calculation of Rf for these standards. Record this measurement in the data table
for pens.

11. place a dot in the center of the darkest area of each circled spot and measure the distance
from the bottom pencil line to the center of each spot. This measurement is the ``Distance
to spot" for each spot. Record each of these measurements in your data table.

12. When finished, dispose of both solutions in the waste container in the lab.

13. Staple your chromatograms to the data sheet below.

Chromatoclraphic Data usinci water (you may not need all of the lines

Pen 1: Distance to solvent front Chromatogram

Spot 1: Distance to spot co,orofspotEL

Spot 2: Distance to spot 4`4 cm co,orofspotfty
Spot 3: Distancv¥`to spot +.G cm Color of spot

Spot 4: Distanc&to spot 4`q __cm Color of spot

Spot 5: Distance to spot

S,.S cm Colorof spot±~
Pen 2: Distance to solvent front

Spot 1: Distance to spot Color of spot

c__==E_ e-c.-__ i

Spot 2: Distance to spot 4.n- cm Colorof spot Y~`\oJ7 `^f ,c€cn , 6di

Spot 3: Distance to spot

4,i color of spot orrty_,

Spot 4: Distance to spot Cm Color of spot

Spot5 Distancetospot "/A cm Colorofspot

a
34
ChromatociraDhic data usinc] Nacl solution (You mav not need all of the lines

Pen 1: Distancetosolventfront 7-C) cm Chromatogram

__

Spot 1: Distance to spot

a-_ 6= \,
Cm Color of spot
_-,_-_---==-i--
A

2-i
Spot 2: Distance to spot Cm Colorofspot °r-`7.

Spot 3: Distance to spot Cm Color of spot

Spot 4: Distance to spot Cm Color of spot

S- Eili
Spot 5: Distance to spot cm Color of spot

Pen 2: Distance to solvent front

70 Cm

Spot 1: Distance to spot c),4 Color of spot f>,nv`

Spot 2: Distance to spot

\.i cm Color of spot

Spot 3: Distance to spot

2.I, cm Color of spot

Spot 4: Distance to spot cm Color of spot

trc^ .

Spct5.rjistHneto spor^ A!JA-:in Color of spelJ!JJL

35
 Experiment 2: Data Rubric (20pts)

--, Points
Data are neat and legible 5pts

Significant figures (>80a/o correct) 3pts

Units (>80°/a correct) 2pts

All data are present and make sense 10pts

Deductions (slidina scale based on TA discretion

Lab area left unclean -20pt5

Improper waste disposal -20pts

Disruptive behavior -20pts

Data sheet is missing TA Signature -20pts

Other:

Comments:

Grade for Data Sheet

36
- Experiment 2: Calculations

(The calculations are NOT allowed on the concept review, so make sure you
fully understand them. Keep jn lab manual to use to study for the lab practical.)

You must be able to perform these calculations on your concept review.

Determination of Rf of Chromatographic Spot

Perform the following calculation for each spot jn each chromatogram. Record the Rf values in
the appropriate table in the results section.

th-b`'/c> Phas{., Rf = Distance to center of si]ot (cm) ol +l:`e-i>L-i-.`

iil HvvO

S'.I 1 :
:..: :`-
i.+c-
Distance to solvent front (cm)

I-a,Jil€ Afro,S,
---------------- ` ' N.C,I

r,£y',5J±,`.1+ i c`:`. I s± I y fr _:a.IL.

3lJ.+ I.`-
7 .0 c.-
--.I.+.'vh

a.±±:± = 0 . = ,± --` Af -- C) . = /
7`\ .. = °` fo|€ Af = 4\.u 7 .a <r?
S I.i , ``
t a c-„ S C-* S .`- 5f2J2fc» = a , +2L. 3_fry A/ = C) .q: 3
7` \ cm = 0. G±= £ nf= O,Gr 7 .C)cfro
Sp.++.` 4 . qc^ St2.+ +``
f:±==-_tl-z,,_S-gfty=c,.&J
0 S f`+ sl
---0-LCJ19 = ft= 0.b q7,1ch

S=:=o.T1_+~~fty=D]T]7`1c-
<,

=fed s ..
7- a ` w+

C=:i:i_-L>.79_5-~~l€f=0.7P
7-0a-
RA sL£,.. tf#"--o.sb=rty<fif=o,sG7.Icn i?4h" SL£L'.. Ci'Lzi== = a.oJL|¥/74=4,Ot°7.c>€-

SL# f#L^=o.„±rty~Af=d.$77`/c.in 5fo,2-.-

/:===-_O,/T±~~f!f`=O-177`z)a-

`po.+3`. 43=t:,_D.Lo±gA/~-d,G/7`1c- 'sL=1L=, 2--±|i=--a.3/_4--_Jay:=0.31

?. C) < try

5±+=>"32±=rty_-D.b37.I`-- 5rbl..i: > `3 i~ ~ a . +Lit = 4 i d++77.c>-

®
39
Experiment 2: Results Table
(Allowed on concept review, pass in with your concept review & data sheets)
'Lr2_cL D ateF!JJ±!r sectl om
Name: c*-.,L2-cl
All results must be written in pen. Pencil is not allowed!!
Record all results with the correct number of significant figures and units.
No marks or notes should be present on this page. Only the tabulated results are
allowed.

ChromatoaraDhic Data usina water

Pen 1 color: Plc/wr` Pen 2 Color:

Penl Rf from Pen2 Rf from

spots Color chromatogram spots Color chrormtogram

1 yc,I/a, 0. s_/ 1 f?.fty%

C) . 5-b

2 0/;...,- C). 4, 2,
2 Yt ,' a J 4/, S-1
V,Lcar
3 0 ` c =- 3 Dr4- C). G I

4 Pu,`,J(utl c) . c c7 4 fry (-` . C a

5 C5,ot~ Crty- C) . 77 5

6 6

ChromatoaraDhic data usina 5% NacI

Pen lcolor: Csrc.~~ Pen 2 color: fry

Pen1 Rf from Pen2 Rffrom
spots Color chromatogram spots Color chromatogram

1 y<''o- D,/G 1 f.`b EL C).c)/a

2 0'_- a,=/ 2 `Ill9Ult a.IJ
3 rLJ C) . i- 3 3 c)r-ul CJ, 3 /

4 P.r,L I ,`.`d'`.Q. C) . Q' 4 4fe(`` CJ ` i- J

5.
•¢=.-.`-:..+ C>. 7q 5
-,

6 6

37
Experiment 2: Results Table Rubric (20pts)
Points
Tables are neat and legible 5pts

Significant figures (>80% correct) 3pts

Units (>80% correct) 2pts

All results are present and make sense 10pts

Deductions (slidina based on TA discretion

Results to riot match data -20pts

Plagiarism!!! Results are identical to another student -100pts

Other:

Comments:

Grade for Results Table

38
 CONCLUSION / ANALYSIS: Since the stationary phase has roughly the same polarity as
water, I hypothesized that the best separation pattern of the dye mixtures would be achieved by
using a more polar solvent (5% Nacl solution) in the mobile phase. Additionally, since the 5%
Nacl solution is more polar than the distilled water, this means it is also more polar than the
stationary phase, and so I anticipated that the 5% Nacl solution would allow the dye mixture's
more-polar molecules to separate from the less-polar molecules within the entire length of the
chromatogram.
My hypothesis was correct, and ideal separation was achieved using the 5% NacI
solution. The chromatography experiment showed that the brown dye from pen 1 is a mixture of
five components (yellow, orange, red, purple, and blue), and the red dye from pen 2 is a mixture
of four components Oink, yellow, orange, and red). From the results of both chromatograms and
the calculated retention factor (Rf) values, I concluded that (a) pen I and pen 2 were more
soluble in the distilled water mobile phase and less soluble in the 5% Nacl solution mobile phase
due to minor dye separation and notably all the components from both pen 1 and pen 2 traveled
near the solvent front line in the distilled water mobile phase, (b) the more polar solvent (5%
Nacl solution) mobile phase allowed the individual components of pen 1 and pen 2 to separate
ideally and adhere along the stationary phase at varying lengths, which clearly showed each
component's level of polarity, solubility, and speed of travel in the mobile phase. By comparing
the Rf values for pen 1 in the distilled water mobile phase to the 50/o Nacl solution mobile phase,
the decrease in Rf value for each component (except for blue) indicates the that components are
significantly less polar, less soluble, and travel much slower while in the 5% Nacl solution
mobile phase. The sane indications are shown by comparing Rf values for pen 2 in the distilled
water mobile phase to the 5% Nacl solution mobile phase. The Rf values are much closer to
each other for the chromatogran using distilled water for the mobile phase, whereas the Rf
values are nicely spread out for the chromatogram using 5% Nacl solution for the mobile phase.
When trying to separate and identify unknown mixtures of dye, it may be necessary to complete
several trials of chromatography runs using solvents of varying polarity until the best level of
separation is achieved.
I believe I did not encounter any sources of error throughout my chromatography runs.
However, if care is not taken while performing paper chromatography experiments, possible
sources of error may include: using pen to mark the chromatography paper instead of pencil Gen
ink can diffuse into dye mixture, and/or obscure the solvent start line), dirty hands and fingers
can contaminate the chromatography paper by leaving residues such as dirt and oils, placing the
chromatography paper in the beaker and allowing the solvent start line/dye mark to become
submerged by the mobile phase (dye will bleed into the solvent and skew the results), leaning the
chromatography paper against the beaker (the paper stationary phase should be standing upright
by itself or hanging down from a clip to avoid this), marking the chromatogram while it is still
wet/danp (this may cause rips or tears in the chromatogram).