Christopher Aretz
Jason Maciolek
CONTINOUS SEPARATION OF JUICE PIGMENTS
Introduction:
This experiment focuses on the continuous separation of pigments from organic fruit juices. Juice pigments are used in various types of natural food dyes. Juice pigments can be separated by a process known as Solid Phase Extraction (SPE). SPE is traditionally done with one column and has four basic steps: equilibrating, loading, washing, and rinsing the column.
This can be a time consuming process when it requires that large amounts of material be separated. With the use of the Semba® Biosciences Octave 100, the SembaPro script software, and its valve block working together these instruments are able to run and control up to eight columns simultaneously. The Octave 100 allows for flexibility in running different methods of liquid chromatography. Because of this we were able to create a new continuous SPE method, separating target pigments from organic fruit juices. This application represents a novel use for the instrument beyond its primary design. This method would utilize four columns; each column being in a different stage of separation and rotating through the stages. This rotation of stages effectively allows for continuous separation of pigments from non-pigment compounds, like sugars, in organic juices. Using a continuous process can free up time and allow for large amounts of juice to be separated.
Materials and Methods:
The Octave 100 made by Semba
®
Bioscience was used as the backbone of this project. A sequence was programmed into the software. This sequence gave the instrument the specific instructions of how and when to switch between different solvents, juice to be filtered, the waste output, and raffinate output.
Filtration methods:
Celite Method: About 3g of Celite 521 was added to the juice and stirred for 10 minutes at room temperature. This mixture was allowed to settle for about 5 minutes. This mixture of Celite 521 and juice was then vacuum filtered through a bed of Celite 521, about 0.5 cm thick, on top of a
Whatman #1 filter paper. The filtration rate appears consistent over time and clogging doesn't seem to be a problem. Filter diameter was 4.25cm, but could easily be increased for faster filtration. The filtrate appeared clear. To check on the performance, this solution was filtered through a 0.45µm Millicup HV, PVDF membrane with a 300 mL capacity and 11.5 cm
2 capacity. The juice flowed through this cup rapidly, without clogging, and only left a minor stain on the filter membrane. This stain could be removed by running water and acetone through the filter cup.
Improved Juice Method: For juices that the above method didn't work for, namely grape juice, this method could be used to allow for filtration of said juices. Also, 3g of Celite 521 could be added to the juice and stirred for 10 minutes at room temperature. If that is done, filter the juice, once it settles, through a bed of Celite 521 on a Whatman #1 filter paper. This won't need to be

done on every juice. This juice, filtered with or without the Celite 521 filtration method, then was filtered through the ChemGlass filtration device that allows for the Millipore filter disks, of different sizes to vary the filtration flow, to be inserted into the device. This paper allows for switching of filter papers easily. This method requires filtering through a larger filter paper first than a smaller one second.
Cranberry Juice:
Ocean Spray 90MX spray-dried cranberry powder, 19.0g, was with D.I. water, 729mL, for 10 minutes at room temperature. This juice mixture went through filtration process. Either of the filtration methods above could be used, but the second one was the faster method.
Other Juices:
Other juices used in this project were purchased at a store. These juices had to be organic juices.
Continuous SPE steps:
Table 1 was programmed into the Octave 100 to allow for the script to run. This script would direct the instrument and allow for separation. This script would need to be varied between different juices. Some juices will need more water pushed through the columns in the wash step than other juices. Table 2 describes each step in detail. Table 3 shows the output of each step in the sequence. Table 4 describes each of the steps of the output in detail.
Table 1: Steps programmed into the Octave 100 software
Step #
1
2
3
4
Column 1
Condition
Column
Feed juice
Water wash
MeOH/EtOAc wash
Table 2: Details about each step
Condition Column
Column 2
MeOH/EtOAc wash
Condition
Column
Feed juice
Water wash
Column 3
Water wash
MeOH/EtOAc wash
Condition
Column
Feed juice
Column 4
Feed juice
Water wash
MeOH/EtOAc wash
Condition
Column
Feed Juice
Column is conditioned with water, allows for each column to be in the same condition and be ready for Feed. Removes any MeOH/EtOAc still in the column.
Filtered juices are injected into the columns to be separated
Water Wash
MeOH/EtOAc wash
Removes everything but the pigments from the juice. Pigments will stay in the column.
(Amount needed may vary between different types of juices.)
Removes everything that sticks the column from the column. The pigments should be

removed here and collected.
Table 3: Output of the Script.
Step # Column 1
1
2
Waste
Waste
3
4
Waste
Raffinate
Table 4: Description of each output
Raffinate
Column 2
Raffinate
Waste
Waste
Waste
Column 3
Waste
Raffinate
Waste
Waste
Column 4
Waste
Waste
Raffinate
Waste
Waste
Pigment should exit the column to be collected. Port F on the value block
Waste consists of water, non-pigment compound from the juices. Can go down the drain
Results and Conclusions:
Table 5: Starting, Ending and Percentage of juice pigments
Juice Starting Mass Ending Mass Percent of Starting
Mass
Cranberry Run 1 0.6717g
Cranberry Run 2 0.6717g
0.0727g
0.0586g
10.82%
8.72%
Blueberry 33.81g* 1.209g 3.046%
Cranberry and Blueberry : In the table above the masses of cranberry and blueberry powder was taken. After running the juices through the continues SPE method the ending masses were taken and the resulting percentages were calculated; this mass should be mostly pigments. The percentages of the starting masses are typical of the percent of pigments that are in fruit juices.
Through other research, cranberry has shown to have ~6% of the mass being pigments.
The data above implies that the pigments of cranberry juice are being separated from the juices, but the separation of these pigments isn’t perfect yet. Further method development is needed to insure the complete or nearly complete separation of pigments from the cranberry juice. The data of the blueberry juice shows that the method developed for separating pigments from the blueberry juice was a success. No more method development is needed for that specific juice. Further work on the method development in combination with the Semba® Bioscience
Octave 100 instrument and software will allow other pigments to be separated from other organic juices.
Setbacks :The computer that controls the Semba® Bioscience Octave 100 instrument malfunction and was not able to communicate with the Octave 100 anymore. Due to this setback no other juices were run through continuous solid phase extraction experiments.

The rest of the juices are filtered and ready to be put through the Octave 100 once it is up and running. Tart cherry, grape and pomegranate are the juices that are left to be put through the machine.