14 Experiment 8: Water Analysis and an Introduction to Chromatography

Great video about chromatography: https://elearning.cpp.edu/learning-objects/organic-chemistry/tlc/

https://chemistrytalk.org/20thin-layer-chromatography/

OBJECTIVE
1. To separate pigments in black ink, to separate the ions in a water sample, and to determine the total ion content and water hardness of that water sample.

CONCEPTS APPLIED
1. Chromatography – one of the most important and powerful separation techniques currently available.
2. Charge balance of aqueous solutions

INTRODUCTION
Chromatography is a technique that separates1 components of a mixture based on differences in their relative affinities (intermolecular forces) for two different materials. The word chromatography comes from Greek words meaning “color” and “to write.” The analyte sample is dissolved in a solvent, called the eluent or mobile phase. The eluent flows over/through a solid material that is called the stationary phase. The stationary phase is commonly made of cellulose (paper), silica or alumina; all of which can be and frequently are chemically modified to have a particular effect on the separation. Depending on the type of chromatography, the solvent can be a liquid or gas and the stationary phase can be in the form of a thin sheet or column. The column can either be packed or coated on its inner surface with the stationary phase material.
In general, each component of the mixture does not stay permanently in either the mobile or stationary phases, but instead is exchanged back and forth repeatedly during the separation forming an equilibrium between the two phases. The different affinities (attraction) of each component for the mobile and stationary phases reflect their equilibrium concentrations. A component with greater affinity for the mobile phase will have a higher mobile phase equilibrium concentration, spend more time in the mobile phase over the course of the separation
and as a result will move through/over the stationary phase rapidly.

The two types of chromatography used in this project are paper chromatography and a form of high performance liquid chromatography (HPLC) called ion chromatography (IC). Paper chromatography is a very simple and inexpensive separation technique that uses paper as the stationary phase. The eluent (a liquid) is allowed to slowly soak up the surface of the paper stationary phase. As it does, the sample components move and separate based on their different affinities for the eluent and paper. The movement of each component is quantified by the ratio of distance traveled by the substance to the distance traveled by the solvent and this ratio is called the retention factor, Rf. This measurement is a primary means of qualitatively identifying compounds separated by paper chromatography. In ion chromatography, the stationary phase is a column typically packed with a synthetic, organic polymer. The eluent is generally an aqueous solution containing small amounts of specific acids or bases, such as nitric acid or sodium bicarbonate, and possibly some small amounts of organic solvents such as acetone or methanol. Completely controlled by a computer, the modern IC instrument consists of a sample injector, pump, column and detector. The most important applications of IC today center on the routine analysis of aqueous systems:

a review of basic separation techniques see pages 12-13 in the 10th ed. of Brown, LeMay and Bursten, Chemistry: The Central Science, Prentice Hall, 2006.

drinking water, environmental water systems (ponds, rivers, aquifers, etc.) and industrial processes that require ultra-pure water such as that found in nuclear power plants and semiconductor manufacturing.2

Pigments in Ink – Ink markers are available in a variety of colors and forms: permanent, dry erase, washable, etc. Many of these markers, especially the black ones, use more than one pigment to achieve their color. For instance, the ink in a black marker may actually contain green, purple, orange and pink pigments. As a point of reference, if enough different colors of paint are mixed together, the mixture eventually turns a gray or blackish color. Permanent markers are not supposed to run or smear, but being permanent really only applies to water as the solvent. The ink in these markers is much less permanent when a different solvent is used, such as ethanol or acetone.

Ions in Water – Water normally contains a variety of ions also referred to as minerals, electrolytes, salts, or minerals. Water naturally dissolves these as it comes into contact with soil and rock. Among the most important salts are the chlorides, sulfates and hydrogen carbonates of calcium, magnesium and sodium. Calcium and magnesium are the most prevalent ions that contribute to water hardness, which is actually a measure of the total concentration of all divalent cations. Water hardness is normally expressed as the equivalent number of milligrams of CaCO3 per liter, as shown in Example 1. Water containing less than an equivalent of 60 mg CaCO3 per liter is considered ‘soft’ and water with more than 270 mg/L is considered ‘hard.’3 (Another common unit used to express small concentrations is parts per million (ppm) which is a measure of mg of solute per kg of solution.) Hard water is a problem for some household and industrial uses.

For instance, the hard water ions react with soap to form a precipitate. This results in the need for additional soap to achieve the same cleaning effectiveness. Another important ion in drinking water is fluoride, which helps to prevent tooth decay. Naturally occurring amounts of fluoride are supplemented to achieve a total fluoride concentration of approximately 1 ppm.4 Naturally occurring fluoride levels in the Omaha area are described in MUD’s annual water quality report.4 Unfortunately, certain types of home treatment devices will remove 85 to more than 95% of all the minerals in water, including fluoride. These are reverse osmosis, distillation units and deionization units. Water softeners, however, do not remove appreciable amounts of fluoride. Other common ions present in water are sodium, chloride, nitrate, sulfate and bicarbonate. The presence of these ions in drinking water is generally not a health concern, except nitrate, but do affect its taste. Although the common ions in drinking water are colorless, they can be separated based on the same fundamental principles as the separation of pigments in the ink pens. Different stationary and mobile phases are required and the instrument detects the separated ions based on changes in conductivity of the mobile phase. In paper chromatography, it is more difficult to quantify the amount of a separated substance; however, with the ion chromatography instrument and computer, the area under the peak is quickly measured and is directly
proportional to the quantity of substance present.
2 Eith, C.; Kolb, M.; Seubert, A. Practical Ion Chromatography; Metrohm Ltd.: Herisau, Switzerland, 2001-02.
3 Harris, D. C. Quantitative Chemical Analysis, 5th ed.; Freeman, 2001.
4 Metropolitan Utilities District; City of Omaha, NE: http://www.mudomaha.com.

Calculation:
Example 1 – Calculate the hardness of water containing 4.62 ppm Mg2+ and 3.74 ppm Ca2+.
(Assume that the density of the solution is 1 kg/L.)

Plan
Step 1. Convert ppm to molarity assuming the density of water is 1 g/mL.
 
= ≈ 1 L solution
1 mg solute
1 kg solution
1 ppm 1 mg solute

Step 2. Add Ca2+ and Mg2+ molarities (retain units of M Ca2+)

Step 3. Convert equivalent M Ca2+ to equivalent mg CaCO3 per liter of solution.

Solution
Step Calculation
1 

 
4.62 mg Mg2+
1 kg solution 

 
1 g Mg2+
1000 mg Mg2+ 

 
1 mol Mg2+
24.30 g Mg2+ 

 
1 kg solution
1 L solution = 1.90 x 10–4 M Mg2+
 
 
3.74 mg Ca2+
1 kg solution 

 
1 g Ca2+
1000 mg Ca2+ 

 
1 mol Ca2+
40.08 g Ca2+ 

 
1 kg solution
1 L solution = 9.33 x 10–5 M Ca2+
2 1.90 x 10–4 M Mg2+ + 9.33 x 10–5 M Ca2+ ≅ 2.83 x 10–4 equivalent M Ca2+
3 2.83 x 10–4 eq. M Ca2+

 
1 mol CaCO3
1 mol Ca2+ 

 
100.0 g CaCO3
1 mol CaCO3 = 2.83 x 10–2 g CaCO3
L solution
2.83 x 10–2 g CaCO3
L solution

 
1000 mg CaCO3
1 g CaCO3 

 
1 L solution
1 kg solution = 28.3 eq. mg CaCO3
1 kg solution
28.3 < 60; therefore, this water is soft.

SAFETY

• Isopropyl alcohol is flammable and may cause eye, respiratory tract and skin irritation
• 5% acetic acid solution (commonly available as vinegar) does not pose any serious health or safety hazards. Information for glacial (~pure) acetic acid is not relevant.
• Carbonic, nitric, sulfuric and dipicolinic acids as used in the ion chromatographs, all are very dilute and therefore do not to pose any serious health or safety hazards. These solutions, after being used, are collected and disposed of by a department professional.

POSSIBLE ADVANCE PREPARATION… ONLY IF INSTRUCTED IN LAB
• In the previous week’s lab, acquire an approved, properly labeled test tube with a lid from your laboratory instructor to use to collect your drinking water sample. Determine who will acquire the water sample and from where. (You will only need one water sample between you and your partner.)
• Record the date, time and any other identifying information about the sample including water system (i.e. private well, MUD, etc.) and address for tap water or brand, place purchased, and manufacturers id code for bottled water.

PROCEDURE
This experiment is intended to be collaborative. You will be working in pairs throughout these
experiments but will be evaluated based on your individual notebook/report.
1. Paper Chromatography Separation of Dyes
Step 1. Obtain two pieces of chromatography paper.
Step 2. Using a pencil, draw a straight line 1 cm from the bottom
of each piece of paper. On the line, make 4 marks 2 cm
from each other and at least 2 cm from either vertical
edge of the paper. Label them A thru D.
Step 3. With each piece of paper, make a small spot 1-2 mm in diameter with one of the markers at mark ‘A’. Repeat, using a different marker on each remaining mark. Be sure to record which spot corresponds to which marker.
Step 4. Form a cylinder and staple as shown so that the line and spots are at the bottom, making sure that the two stapled edges must not touch, Figure 22.
Step 5. Collect approximately 20 mL of 70% isopropyl alcohol solution in a 400 mL beaker and 20 ml of vinegar in another 400 mL beaker. Make sure that the spots are not submerged in the vinegar/alcohol.
Step 6. Gently place one piece of chromatography paper in each beaker and cover with a watch glass. Be sure that the paper does not touch the sides of the beaker. The eluent will move up the paper by capillary action.
Step 7. After the chromatograms have developed for 15-20 minutes or when separation of the individual ink pigments has occurred, remove the chromatograms from the beakers. (Do not allow the solvent front to get all the way to the top edge of the paper.) Carefully remove the staples and lay flat to dry. Quickly (before the eluent evaporates) draw a line in pencil across the paper at the solvent front.
Step 8. Remaining 70% isopropyl alcohol must be collected in the unwanted materials container. Discard remaining vinegar into the sink with a 5-fold excess water.
Step 9. Keep your chromatograms and submit them with your lab report (one per student).
Step 10. Create a table for each marker and eluent, record the colors separated, their order, their Rf values and any other pertinent information. The Rf value is the principal means of quantifying the behavior of the substances being separated.

Rf= distance from the base line to the center of each color / distance from the base line to the solvent front

2. Ion Chromatography of a Water Sample
You will analyze your water sample twice: once for anions and once for cations. The procedure is the same for both; however, the instruments have been setup differently. (Three instruments will be setup for cation determination and three will be setup for anion determination.) Determine which Eco IC instrument that you should use first. The MagIC Net software should already be open and the workplace window, Figure 23, should be visible. (Check with your lab instructor if this is not to be the case.) The Eco IC instrument, Figure 24, and it’s computer should both be on and ready for your sample when you get there.

Figures for MagIC Net workplace window and Metrohm Eco IC instrument

Step 1. To begin your sample analysis, make sure that the ‘workplace’ tile on the left and ‘single determination’ tab are both be selected as shown in Figure 23.
Step 2. Enter your sample number in the ‘Ident’ field and your initials. Leave the other fields unchanged.
Step 3. Click the green ‘Start’ button, Figure 23.
Step 4. Use a Kimwipe® to wipe clean the last 3-4 inches of the inlet tube, then place it in your water sample. Put the empty syringe in the hole in the front of the instrument and draw approximately 1 ml into the syringe.
Step 5. Click continue to begin the separation/analysis of your water sample.
Step 6. Detach the syringe and empty it into a sink. Remove the inlet tube from your water sample and replace the lid on the sample bottle. Wait for the analysis the analysis to complete – usually about 10 minutes. The screen will reset when it is complete.
Step 7. To print your chromatogram, click the ‘database’ tile on the upper left of the screen. Find your file and select it by clicking on it. Then click ‘file’ (in the far upper left corner), then ‘print’, then ‘report’. Choose your printer and be sure that ‘selected determination’ and NOT ‘all determinations’ is selected.
Step 8. Record in your notebook the information in the table on the bottom of the printed report: ion name, retention times, peak areas, and concentration in mg/L. Leave space for a column on the right where you will later add the concentration.
Step 9. Repeat steps 3-8 for determination of the other ions being sure to use an instrument set up for those ions.
Step 10. Enter your results on the project website water.unomaha.edu.

DATA PROCESSING
1. Record these concentrations on the data collection website before you leave the laboratory.
2. Convert the concentrations to units of molarity and record these to your data tables. (Step 1 in the example) Values less than zero should be considered zero.
3. Check the charge balance of your sample. Just as an ionic solid must maintain electrical neutrality (CaF– is not a stable ionic substance but CaF2 is) so do solutions with dissolved ions. After multiplying the molarity of each ion by the absolute value of its charge, the sum of the cation charge should equal the sum of the anion charge.

For example, if Na+, Mg2+, Cl– and SO􀬸
􀬶􀬿 are the only ions observed, then the following
must be true. [Na+] + 2 [Mg2+] =[Cl-] + 2 [SO􀬸
􀬶􀬿] (sum of the cations = sum of the anions)
where [X] refers to the molarity of species X.
If [Na+] = 2.7610-3 M, [Mg2+] = 7.2010-4 M, [Cl-] = 5.5910-4 M, and [SO􀬸
􀬶􀬿] = 1.5310-3 M
Then 2.7610-3 M + 2(7.2010-4 M) = 5.5910-4 M + 2(1.5310-3 M) + x,
where x is the difference between the cation and anion sums.
In this case, x = 5.8110-4 M, and it most likely represents the bicarbonate ion
concentration.
Note: If x were negative, it would represent a cation concentration.

4. The ions detected in your water sample probably do not achieve a charge balance because one anion, bicarbonate, was not detected by the chromatograph and is therefore not included in your chromatograms. It is not detected because it is also part of the eluent. (Any bicarbonate in your sample is indistinguishable from the bicarbonate in the eluent.) Assuming that your separations went well, bicarbonate constitutes the remaining ions needed to achieve charge balance. Report the difference in your charge balance as the concentration of bicarbonate.
5. Add this ion and its calculated concentration to your data table.
6. Be sure to submit a copy of each chromatogram report with your lab report.

DISCUSSION
Address the following topics in your discussion:
1. Pigments with the smallest Rf values have the greatest affinity for which phase, the mobile or stationary phase?
2. Were there any unidentified ions in your water sample that showed up on your chromatogram? If yes, suggest some other possible ions that might be acceptably found in drinking water, i.e. not CN– (cyanide). (Disregard peaks or dips that appear on your chromatograms prior to sodium or fluoride.)
3. What is the hardness of your water sample in equivalent mg of CaCO3 per liter? (see the example)
4. Does your water appear to be fluorinated? Cite evidence to support your conclusion.

CONCLUSION
Write a conclusion summarizing the main results of the experiment.
EVALUATION
Credit is based on your recorded observations/data, calculations, responses to the pre-lab questions, and discussion/conclusion.

Ron:

THIn-LAYER CHROMATOGRAPHY (tLc) ANALYSIS OF INKS

Objective: to separate and identify components of inks

LEARNING POINTS:

1. Learn how to prepare and spot a TLC plate with samples
2. interpret distance traveled by samples and calculate Rf values

The technique known as thin-layer chromatography (TLC) is a method utilized to separate non-volatile mixtures onto a rigid and solid suppor. Thin-layer chromatography can be accomplished upon a sheet of plastic, glass, or even aluminium foil.  These supporting scaffolds are coated with a thin layer of adsorbent material, commonly a silica gel, aluminium oxide, or cellulose. This absorbent material is referred to as the stationary phase.  The solvent (liquid mixture ascending the absorbent material) is referred to as the mobile phase.

The test analyte is solubilized into a suitable solvent in order for it to be applied to the absorbent material in preparation for analysis.  After the test sample has been applied onto the absorbent material, then the plate is placed into a chamber containing the mobile phase.  Often the chamber is covered or sealed so that the vapors from the mobile phase can equilibrate into the chamber and enhance the ascending of the mixtures and improve separation.   The mobile phase is drawn up the plate by means of  capillary action. Due to the influence of the stationary phase, mobile phase, and characteristics of the analyte there are usually differences in rates of the analytes ascending the TLC plate which results in separation.

Thin-layer chromatography has many uses.  This technique can be applied to monitor the progress of a chemical reaction, identify components present in a given mixture, as well as determine the purity of a substance. Some examples of various types of uses include: separation of fatty acids, identification and separation of pesticides or herbicides in food and water, analysis of dyes, forensics analysis, quality control for purity radiopharmaceuticals, and identification of components comprising medicinal plants.

For example, in organic chemistry, it is possible to qualitatively follow reactions with TLC.  Mixtures to be analyzed are sampled with a capillary tube and spotted onto the plate.  The components of complex mixtures of organic compounds can be examined by TLC.  Prudent choices made concerning the mobile phase composition and the type of stationary phase can aid tremendously the efficiency of test sample separation.

Ink is commonly used substance that exists as a liquid or paste.  Ink will contain dyes or pigments which are used to color a surface producing an image, written text, or an artistic design. A quill, brush, or pen can be utilized for purposes of drawing or writing. For lithographic printing or art, and letterpress will utilize thicker compositions of ink referred to as paste.

The ink itself can be a complex mixture of dyes, pigments, solvents, various resins, solubilizers, various surfactants, and solvents. These components of inks have many functions that may include one or more of the following: carrier of the ink, the colorants themselves, additives to influence the flow and/or thickness of the ink, as well as its appearance when dried.

Formulas of inks will vary widely, however, generally the following four components are present in some manner:  colorants, binders, additives (vary widely), carrier compounds.  Inks will fall into four classes: aqueous, other liquid (e.g. organic solvent), paste, or powder.  For colorants, pigment inks are more common than say for dye-based inks because they are color-fast, more expensive, less consistent in color, and have lesser range of color.

Pigments are solid yet opaque particles suspended in ink to the desired color. Pigment molecules themselves are typically linked together to form a crystalline structure. These crystalline structures comprise 5 % to 30 % of an ink volume.  The type of pigment greatly influences the hue (color or shade), saturation (saturation defines the range from a pure color to grayness at a constant lightness level), and lightness (a range from dark to fully illuminated). A pure color is fully saturated.

A dye-based ink is usually much stronger than pigment-based inks and can produce a great deal more color of a given density. However, there are limitations to dye-based inks because dyes are usually dissolved into a liquid phase, have a tendency to soak into the paper which makes the ink less efficient and allows the ink to bleed at the edges of a desired image.

Safety

Use the normal laboratory precautions identified on the safety sheet you signed.

Some solvents may need to be collected in a waste bottle.

Dispose of paper wick (see below) and used paper towels in trash container.

PROCEDURE

1. Obtain a 400-mL beaker, glass stir rod, prepared TLC plates, and designated ink pens.
2. You will first draw a pencil line (carbon

from pencils do not ascend the TLC) and

carefully draw a thin pencil line approximately

½ inch from the bottom of the TLC plate.

3. Dot the ink of designated pens along the pencil line. The dots need not be big. Making the dots too big can cause blending of dyes/pigments from the pens as the mobile support ascends the TLC plate.
4. Into the 400-mL beaker place distilled water

so that the level of water does NOT touch or

submerge the ink dots along the pencil line.

5. Carefully hang or tape the prepared TLC

plate onto a glass stir rod the carefully place

the TLC plate into this chamber.

6. Allow the mobile phase to climb at least

three-fourths the way up the TLC plate.

7. When completed, carefully remove the

TLC plate from the chamber and stir rod.

Place the still wet TLC plate onto clean paper towel to dry.

Calculation of Rf values:

When the TLC has dried you will obtain a ruler and measure distances traveled by the pigments in order to calculate Rf values.

Rf    =      distance traveled by the compound          =       A

distance traveled by the solvent front                 B

1    2   3   4    Lanes

Exercises:

1. Calculate and report the Rf values of all pigment components of each pen you tested. You can place the results into a table.
2. Count the number of pigment deposits for each lane in the TLC shown above.
3. How many are in lane 1?
4. How many are in lane 2?  Do any deposits coincide with other lanes?
5. How many are in lane 3?
6. How many in lane 4? Do any deposits coincide with other lanes?
7. Which pen of the all you tested by the TLC had the most colored substances?
8. Which pens had colored substances in common?
9. Which pens had only one colored substance?