Omar Alkhadra Chem 2 lab

Prof. Kipruto

Introduction:

In this lab experiment liquid chromatography will be used to separate the components found in grape-
flavored Kool-Aid. The main mechanism that is used to separate the components of Kool-Aid is the
polarity of the substances. There are two phases that allow the mixture to get separated, stationary and
mobile phases. The stationary phase is what will attract the substances in the mixture separating from
the liquid and keeping it in the chromatography column. And the mobile phase is what will individually
separate the components by attracting them according to their polarity.

Materials:

 C18 Sep-Pac cartridge

 10 ml syringe with male luer tip
 1 ml syringe with male luer tip
 Two 10 ml graduated cylinders
 Two 25 ml graduated cylinders
 70% isopropanol
 Grape Kool-Aid
 Distilled water
 Four 50 ml beakers
 Three 100 ml beakers

Methods:

There are two parts to this lab, for the purposes of this lab we will only concerned with the first part.

The purpose of this experiment is to use liquid chromatography to separate the component substances
that are contained in grape-flavored Kool-Aid. The first step is to grab all the materials needed to get the
experiment started. Then the first solvent, the mobile phase, is prepared by mixing 13 ml of 70%
isopropanol with 27 ml of distilled water to make 18% (v/v) isopropanol solution. Next the C18 Sep-Pac is
is prepared, first by cutting the tips off of it then by injecting 10ml of 70% isopropanol through at a rate
of 5-10 ml per minute then washing by injection another 10ml of distilled water through it. Next 1 ml of
the Kool-Aid mixture is injected through the Sep-Pac and it can be seen how the colors of the
components of the Kool-Aid are attracted to the stationary phase. The effluent that washes out of the
column is collected and discarded. The next step is where the data collection starts. A 10ml syringe is
filled with the 18% isopropanol solution prepared earlier. The solution is then slowly injected into the
Sep-Pac and the eluent is collected into a 10ml graduated cylinder. How much of the aluent washes out
into the graduated cylinder is recorded as the first and last drop of red and blue are extracted from the
Sep-Pac into the cylinder. Wash the Sep-Pac with 70% isopropanol and distilled water and repeat the
procedure 3 times and record all the data.

Results and calculations:

Red Blue
Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3
Vr (Start) ml 0.9 0.9 2 1.3 2.1 2.9
Vr (End) ml 1.2 1.9 2.8 3.1 3.0 4.0
Omar Alkhadra Chem 2 lab
Prof. Kipruto

For part one the selectivity and the separation need to calculated using their respective formulas. To do
those calculation the Bandwidth (W) of each trial of both colors needs to be calculated and the Wavg is
taken.

The red dye

 W1 = 1.2 – 0.9 = 0.3

 W2 = 1.9 – 0.9 = 1.0
 W3 = 2.8 – 2.0 = 0.8

Wavg = (0.3+1.0+0.8)/3= 0.7

The blue dye

 W1 = 3.1 – 1.3 = 0.8

 W2 = 3.0 – 2.1 = 0.9
 W3 = 4.0 – 2.9 = 1.1

Wavg = (0.8+0.9+1.1)/3= 0.93

Now Calculate the average retention

First the starting avg is calculated by adding the values recorded for the three trails and divided by 3
which gives 1.2 for Red and 2.1 for Blue. If the starting avg is not calculated, and the avg retention was
calculated for each trail, the result from all 3 trials would have to be averaged out get the avg for the
entire color and not just one trial of the color. Those numbers are plugged in the formula given above to
get average retention.

0.7
Ravg =¿ 1.2+ =1.55 for the
2
v¿
0.93
Ravg =¿ 2.1+ =2.65 for the
2
v¿
Next using the Vm ( Vm=0.5 π r 2 L¿ which gives 0.49 ml, Capacity factor (k’) can be calculated using
the following formula

Vravg−Vm
k'=
Vm
1.55−0.49
k'= =2.163 for the ¿
0.49
Omar Alkhadra Chem 2 lab
Prof. Kipruto

2.65−0.49
k'= =4.408 for the¿
0.49
Next the separation factor (α) is calculated
'
k
'
k
4.408
α =¿ ¿¿ ¿= =2.037
2.163
Finally, the resolution of separation (R) is calculated

2 ( 2.65−1.55 )
R= =1.34
0.7+0.93
The theoretical plates are calculated to show how efficient the chromatography column is
2 2
Vr 1.55
N=16 ( ) ( )
W
=16
0.7
=78.44 for ¿

2 2
Vr 2.65
N=16 ( ) ( )
W
=16
0 .93
=129.9 for ¿

Discussion:

The calculated values that were acquired in the section above of α , k ' , N and R, show that all
values are within their normal range which shows that there was good separation of food dye in Kool-
Aid. This is achieved by the difference in polarity between the mobile phase and the stationary phase.
Looking the general rule that “like dissolves like” that applies to solubility, this tells us the compounds
that have same polarities are more likely to dissolve with one another. Since isopropyl alcohol is different
than water in that it is has hydrogens surrounding giving it very little polarity unlike water, which makes
water much more polar and that allows for the separation of the dyes during the experiment.

Areas where error might have occurred and may have had an impact on the values obtained during the
experiment is not cleaning the tube and cylinders properly between the trails. Not injecting the mixture
through the column at the proper rate. Also not putting a clear white back ground when focusing the
color change of the drops while injecting the Kool-Aid through the column.

Conclusion:

Liquid chromatography allows for seperatinog of dyes in a liquid substance and it can determine the
polarity of the components. Kool-Aid is make up of blue and red dyes which make the purple grape color,
and since the resolution value obtained was grater than one this experiment can be deemed successful
 Omar Alkhadra Chem 2 lab
Prof. Kipruto

Works cited:

1. Libretexts. “Lab 2: High Performance Liquid Chromatography.” Chemistry LibreTexts, Libretexts, 19 Dec.

2016,

chem.libretexts.org/LibreTexts/University_of_California_Davis/UCD_Chem_115_Lab_Manual/Lab_2:_Hi

gh_Performance_Liquid_Chromatography.
2. “Liquid Chromatography - A. Sedano - AP Chemistry Laboratories.” Google Sites,

sites.google.com/a/moreaucatholic.org/ap-chemistry-labs-2011-12/stuff-of-interest/liquid-

chroma#TOC-Post-lab-Analysis.