Biological Aspects of Making a Salad

Nutrition is the outstanding biological consideration which makes salads important. Raw dark green leafy vegetables are superb sources of vitamins A, C, E, (the anti-cancer vitamins) and K, as well as possessing respectable quantities of the B complex. Vitamin C is particularly difficult to obtain in adequate quantities in winter months, making the consumption of salads at that time even more valuable. In addition, one’s susceptibility to colds and infection increases when supplies of C and A are diminished. An excellent supplementary source of fresh salad makings during the winter months are sprouted seeds (alfalfa, broccoli, etc). See a previous handout on sprouting seeds.
Often, a simple salad can be both elegant and delicious especially if made with fresh ingredients. Darker greens such as kale, spinach, arugula, etc, are especially rich. Darker leaved lettuce such as romaine, Bibb, and leaf lettuce are far superior to iceberg lettuce which is nutritionally relatively poor. Garlic is a mainstay for many salads. If you wish to add variety to your salad, you might add one or two of the following: red bell pepper, grated carrots, grated apples, parsley, onion, grated or cubed cheese, or even anchovies. However, combining too many different ingredients can detract from its enjoyment.

A salad is “dressed” with oil and an acid, usually lemon juice or vinegar. Important to proper dressing of a salad is to remember to use relatively dry makings so that the oil will stick to the leaves. After the greens are properly coated with oil, then add the acid, usually in the proportion of 1:2 or 1:3 parts acid to oil. Do not add the acid until shortly before serving since the acid will draw out the liquid from the greens by osmosis, resulting in a wilted salad.

For four salad eaters:
1 medium-large clove garlic minced
1 cup. coarsely chopped, deveined kale, spinach, and/or dark green lettuce.
1 cup fresh alfalfa sprouts
1/3 cup finely chopped parsley
2 carrots grated
1/2 small onion thinly sliced
4 Tbl virgin olive oil (other oils can be used, but the flavor may lack richness)
1 1/2 Tbl fresh lemon juice (2 Tbl wine vinegar or apple vinegar will do.)
1/4 tsp salt
1/2 tsp freshly ground black pepper

1. Rub bowl with garlic, crushing pieces against side of bowl.
2. Add dry greens, distributing the sprouts.
3. Gently distribute carrots throughout salad (do not mash). Add onion, toss gently.
4. Drizzle oil over, toss to completely coat. At this point, the salad can wait several hours in the refrigerator for the last steps.
5. Add lemon juice, salt and pepper, and toss to disperse. Serve as first course for optimum pleasure and digestion.

Some people may miss the sugar found in prepared salad dressings. In that case, you may wish to add additional grated carrots or apples. For a hearty salad, small cheese cubes, ham cubes, bacon, toasted sunflower seeds or other addition can be considered. More elegant salads have relatively few ingredients however. Bon Apetito!

See also: Sprouts, Ester Munroe, Steven Greene Pr., Brattleburo, VT. (1974)

Vitamin C Excretion Problems

Vitamin C Excretion Problems

Remember to set up a table to record and process your titration data as follows:

aliquot size being titrated:________ mL

conversion factor of iodine being used: _________ mg vitamin C/mL iodine

 

Trial I Trial II Trial III
finish:
start: __________ __________ __________
iodine used:

The start for trial II should equal the finish of Trial I, etc (assuming no leakage of titrant between titrations). Once the three flasks are titrated, you average the iodine used by adding them up and dividing by three.

The answers to the problems are at the bottom of the page.

PROBLEMS:

1. Standardization:
1.00 mL aliquots of 1% ascorbic acid were titrated in triplicate with iodine of unknown strength.  Successive readings on the buret were: 12.94, 24.35, 35.73, 46.00 mL.  What is the conversion factor for this iodine?

2. Assay of urine:
10 mL aliquots of urine were titrated in triplicate with iodine having a conversion factor of 0.932 mg vitamin C/mL iodine.  The following are successive readings on the buret:  15.91, 16.08, 16.28 and 16.47 mL.  What is the average amount of vitamin C in the 10 mL aliquot?

3. Assay of orange juice:
5 mL of orange juice was found to contain 2.37 mg of vitamin C.  How much vitamin C is there in 100 mL?

4. Calculation of urine production:
What is the urine production rate per hour if the bladder was voided at 2:30 and 510 mL was collected at 4:30?

5. Calc. of Vit C excretion rate: 
If a person produces urine with 1.43 mg vitamin C/10 mL aliquot, and they produced 167 mL in an hour, what is the excretion rate/hr?

6. Calculation of urine production:
A person who drank a lot of Coke produced 230 mL in 20 minutes.  What was their urinary production rate per hour?

7. Calculation of urine production:
A person voided after breakfast at 8:27 AM.  At 12:15 PM, he produced a total of 396 mL of urine.  What was his hourly rate of urine production?  (Hint: express total time as hours, calculating fraction of hr as # of minutes/60)

8. Calculation of urine production:
A poor A&P student voided her bladder at the beginning of Lab (2:12 PM–she came in late…).  At 4:12 PM, she produced 438 mL of urine.  What is her rate of production of urine per hour?

9. Calc. of Vit C excretion rate: 
The student in the previous question titrated 10 mL of urine from question 8 in triplicate with iodine whose conversion factor was  0.729 mg vitamin C/mL iodine.  She got the following successive numbers: 12.01, 14.79, 17.51 and 20.53 mL.

a: What was the content of vitamin C in 10 mL of her urine?
b. What was the total number of 10 mL aliquots which she produced in an hour?
c: What was the total amount of vitamin C which she excreted in an hour?

(Here is problem 9 worked out if you would like to compare it to your work.)

Answers:

1.  0.907 mg vit C/mL iodine
2.  0.174 mg vit C/10 mL
3.  47.4 mg vit C/100 mL
4.  255 mL/hour
5.  23.9 mg vit C/hr
6.  690 mL/hr
7.  104 mL/hr
8.  219 mL/hr
9a.  2.07 mg vit C/10 mL
9b.  21.9 aliquots
9c.  45.3 mg vit C/hr

Vitamin C Assay Introduction and Reagents

Vitamin C Assay Introduction and Reagents

titrated_sample_P5020018sm
the end point is the appearance of the blue starch-iodide color

Vitamin C (ascorbic acid) is required for the synthesis of collagen and to enable WBC phagocytosis, among others. It also serves as an antioxidant, protecting the body from oxidant carcinogens. It is found especially in fresh fruits and vegetables, but is labile, and deteriorates with storage. We will measure vitamin C first in pure solution (to standardize the assay reagents), then in fruit juice. Finally, as a means of evaluating the amount of vitamin C you are taking in, we will measure the quantity you excrete in one hour in your urine.

We will assay vitamin C by using a “starch-iodine” titration. When iodine is added to a starch solution, it reacts to produce a purple color. However, if there is any vitamin C in the solution, it “neutralizes” the iodine, preventing the formation of the purple color. Thus, the amount of vitamin C present in a solution may be measured by first adding a small amount of acidified starch (called “reaction mix”), and then adding iodine drop wise until the solution turns purple. The amount of vitamin C present will be proportional to the amount of iodine required to turn the solution purple. This drop wise addition of a reagent to a specified endpoint is called a titration.

The solutions required to perform the assay for vitamin C are listed below. While they will be available for you in the lab, you should know the function of each of the ingredients in the titration, and how to make up the solutions if needed.
Here are the reagents needed for the titration:

1)  Reaction Mix:  a dilute solution of starch which is slightly acidified to stabilize the color.
2)  0.1 N Iodine : the stock solution of iodine which must be diluted before using for titrations.
3)  0.01 N Iodine :  titrant for the vitamin C titration
4)  1.000% vitamin C :  a standard solution used to determine the conversion factor of the 0.01 N Iodine.
1)  REACTION MIX, for 1 gallon:

1. Weigh out  1.2 g starch powder
2. Suspend starch powder in  200 mL dH 2O. Bring to boil with constant stirring, taking care not to burn.
3. Fill a clean 1 gallon jug half full with  dH2 O.
4. Add the following to the half filled gallon jug:
a. 47 mL concentrated HCl
(CAUTION, extremely corrosive, use goggles, lab coat and fume hood, rinse out graduated cylinder immediately.)
b. 200 mL starch solution from step 2.
c. q.s. to 1 gallon with  dH2 0, mix thoroughly.
5. Use 10-15 mL of reaction mix (Rxn mix) per titration flask, conveniently added with a repipet.
[for 5 gallons of reaction mix: 6 g starch, 235 mL concentrated HCl]

2)  0.1 N IODINE stock solution

1. Weigh out and dissolve in 100 mL dH 2O with much stirring:
12.7 g Iodine
20.0 g KI (potassium iodide)
2.  q.s. to 1 liter with  dH2 0, keep well capped with iodine-resistant plastic-lined lid.

3)  0.01 N IODINE solution (for titrations)
1.  378 mL 0.1 N Iodine added to gallon jug
2.  q.s. to 1 gallon with  dH2 0, mix thoroughly, keep well capped.

4)  1.000% ASCORBIC ACID standard solution:

1. Carefully weigh out  1.000 g ascorbic acid, add to 100 mL volumetric flask
2. Add approximately 50 mL room temperature  dH 2 0, swirl to dissolve.
3.  q.s. to 100.00 mL with room temperature  dH2 0
4. Store capped at 4C.

Determination of Vitamin C Excretion Rate

Determination of Vitamin C Excretion Rate

See  VITAMIN C TITRATION PROTOCOL for detailed directions on how to titrate for the quantity of vitamin C in a sample.  The same titration technique is use to measure vitamin C in aliquots of urine.  The principle is to completely void your bladder and drink a full 12 ounces of water at time zero.  At exactly one hour later, collect 100% of the urine produced during the preceeding hour, measure and record the volume, and titrate 10 mL aliqouts  to determine the amount of vitamin C in 10 mL.  When you multiply the vitamin C in 10 mL times the number of 10 mL aliquots in your one hour sample, you get the hourly excretion rate of vitamin C.  Compare your excretion rate with those of your class mates.  Look for correlations between diet, smoking habits, health, etc for possible explanations of high or low excretion rates.

Here is a Vitamin C Excretion Rate data sheet into which you should enter your data.  It will guide you through the steps and calculations.

Here are class data collected  which indicated a median excretion rate of 10.9 mg vitamin C/hour.

EQUIPMENT:
buret
buret clamp
ring stand
250 mL beaker
three 250 mL flasks
graduated cylinder (100-500 mL, depending…)
urine hydrometer and cylinder
10 mL pipets

SUPPLIES:
standardized 0.01 N iodine
(in 500 mL flasks)
funnel
Starch-HCl Rxn mix in repipet
freshly collected urine
white scrap paper

Here is a pdf summarizing the interpretation of urinalysis and the list of activities which you are to perform to measure Vitamin C Excretion Rate. Here is a sample data page from a recent quiz.

1. Completely void the bladder at the beginning of the Lab, note the exact time to the minute.

2. Consume 12 full ounces of water (soft drink is OK, but it should not be caffeinated). (less and you may

3. Exactly one hour later, collect and measure all urine produced by completely voiding into a 400 mL beaker (more for some…).

4. Record the total volume produced.

5. Measure the specific gravity of your urine using the hydrometer. Note that it should be between 1.000 and 1.040. (Make an illustration.)
DO NOT DROP THE HYDROMETER INTO A DRY CYLINDER, THEY BREAK!
Here are a variety images of hydrometers:
Hydrometer out of its cylinder
Hydrometer reading 1.015 in sugar solution
Hydrometer reading 1.022 in urine
After recording the specific gravity, dip a urinalysis “Multistix 10 SG” strip into the urine, allow to sit for the prescribed time, and score and record the results based on this color chart.

6. Titrate 10 mL aliquots of the urine in triplicate, carefully recording start and finish volumes for each, as given in the titration protocol.

7. Determine the average mL iodine required per 10 mL of urine, multiply times the conversion factor to yield the amount of vitamin C in the 10 mL aliquot.

8. Determine the number of aliquots produced per hour by dividing the total volume of urine produced in an hour by the aliquot size (10 mL).

9. Determine the total vitamin C excreted per hour by multiplying the number of aliquots in the hour’s sample by the Vitamin C per aliquot.

excretion_formula

I.  FILL THE BURET

II.  PREPARE THE SAMPLE:
1. Place 10 mL of reaction mix in 250 mL flask, using a repipet.
2. Pipet in a 10 mL aliquot of urine into each of three flasks.

III.  TITRATE THE SAMPLE:
1. Follow the format for recording your data as demonstrated:  Record the starting volume in the buret to nearest hundredth of a mL (remember that the graduations go from top to bottom of buret, the numbers increases as you go down, and that you read at the bottom of the meniscus).  Before you begin each titration, judge whether you have enough titrant in the buret to finish the flask.  When in doubt, refill the buret, and record the new start reading.
2. Place prepared flask of sample under spout.
3. Add titrant while simultaneously swirling the flask.  Be cautious on the first flask, as it may take much less titrant than you anticipate.
4. When the color change begins to show while swirling, reduce the rate of titrant addition.  Continue to reduce the rate as you approach the endpoint (color will take longer to disappear).
5. Begin adding titrant drop-wise, swirling to remove color after each addition.
6. Stop when a trace of blue is stable (hopefully after a single drop has been added).  The blue color may fade after 10-15 seconds.  Not to worry.
7. Record the finish buret reading to nearest hundredth mL.
8. Repeat this process for the second and third flask, using the finish reading of the previous flask for the start of the current flask.

IV.  CALCULATE THE RESULTS:
1. Determine the mL titrant used for each flask, determine the mean and mean deviation for the three.  (If the deviation is greater than 3%, consider repeating the titration.)
2. Calculate the mean mg of vitamin C in 10 mL urine by multiplying the mean mL titrant used times the conversion factor for the iodine titrant used.
3. Calculate the mg vitamin C/hour multiplying the mg vitamin C/10 mL X (total mL urine produced in an hour)/10 mL aliquot volume.

Titration of Vitamin C in Fruit Juice

Titration of Vitamin C in Fruit Juice

RELATED PROTOCOLS:
Vitamin C Assay Introduction and Reagents
Titration Protocol
Standardization of 0.01 N Iodine Titrant

SUPPLIES:

  • Fruit juice (orange, grapefruit, lemons, etc) and/or prepared juices of other fruits or vegetables
  • Apple, tomato, carrot, orange soda, etc.
  • Reaction mix in 10 mL repipet (see previous protocol)
  • Standardized 0.01 N iodine in 500 mL flask with funnel

EQUIPMENT:

  • Four 250 mL Erlenmeyer flasks
  • 50 mL buret in buret stand on a white piece of scrap paper
  • Fine strainer (if the juice is to be prepared from fruit) funnel
  • 10 mL pipets or 5 mL displacement pipets with tips

PROTOCOL:

1. Prepared juices may be used directly unless they have excessive pulp in them. For pulpy juices (such as citrus), squeeze approximately 40 mL of juice, filter through a fine strainer supported in a funnel into a 250 mL Erlenmeyer flask.
10 mL reaction mix08_10_ml_RxnMx_P5064172

2. Prepare three 250 mL Erlenmeyer flasks by repipetting 15 mL of reaction mix into each. (A 10 mL repipet may be set on 7.5 mL, and add two “squirts.” (It takes more reaction mix than clear specimens to show the endpoint more clearly.)

ready_to_titrate_P5020016
Ready to titrate 10 mL of fruit juice

3. Pipette 10.0 mL of the juice to be tested into each of the three prepared flasks.
titrating fruit juice finished titration

4. Titrate as in the Titration Protocol . Since the juice is colored, the endpoint will be less obvious than with clear solutions, but a distinct bluish darkening will be seen (often a greenish color).
NOTE: The titrated juice may lose its blue upon sitting, but if it is bluish for 5 – 10 seconds, ignore any loss of color after that.

5. Calculate the # mg of vitamin C in 100 mL of juice as follows:
mean mL iodine x conversion factor x 10 = mg vit. C/100 mL.

6. Since 1 mL of fruit juice is very close to 1 g, the concentration of vitamin C in 100 g of the fruit is essentially equal that in the juice. Enter your data into the class data sheet in the computer express as mg/100 g fruit). Be certain to get a copy of the class data sheet for your notebook. Compare these data with the “book value” for these fruits.

Vitamin C Titration Protocol

Vitamin C Titration Protocol

Vitamin C titration protocolThe amount of vitamin C can be determined using a classic starch-iodine titration.  Iodine reacts with starch to make an intense blue-black color, but vitamin C reduces iodine to iodide, thus preventing it from creating the blue color.  When a known volume of solution containing vitamin C is combined with a small quantity of starch/dilute HCl, dropwise addition of iodine will not cause a color change as long as any vitamin C is present.  However, as soon as the vitamin C has been exhausted, the iodine will be free to react with the starch, and a blue color will appear.  The amount of vitamin C present will be proportional to the amount of iodine required to bring about the appearance of the blue color.  If the iodine has been standardized so that the amount required to titrate a known amount of vitamin C, then the amount of vitamin C present in the unknown can be determined.

If you need practice reading burets (i.e., interpolating between marking lines), here is a page of buret images to read.

EQUIPMENT:
buret supported in a
buret clamp and
ring stand
250 mL beaker
three 250 mL flasks
10 mL pipets

SUPPLIES:
scrap white paper
standardized 0.01 N iodine solution
in 250 mL flasks with a funnel
Starch-HCl Reaction mix in repipet
soln. or suspension of sample

I. FILL THE BURET:

02_5_beaker_buret_detail_P5064163

1. Place 250 mL beaker under spout to catch excess iodine solution.
close the stopcock

OLYMPUS DIGITAL CAMERA
2. VERY IMPORTANT: Close stopcock. Otherwise, iodine will flow out all over your bench as you try to fill the buret.

04_fill_buret_P5064166
3. Place a funnel in the buret, and fill with 0.01 N by adding iodine titrant. Add iodine slowly enough so that the funnel is nearly empty at all times. Do not fill up the funnel or the buret may over fill and spill iodine every where…

4. Stop adding iodine solution when the buret is filled to close to the top of the graduations. (Do not try to fill to the 0.00 mL.)

5. Shoot several streams of titrant (iodine solution) to drive bubbles out of the buret spout. (If bubbles reappear in the spout, grease the joint between the valve and the spout with stopcock grease.) Practice controlling the stopcock so that a single drop is added to the flask.

II. PREPARE THE SAMPLE:

1. Place the desired quantity of reaction mix in 250 mL flask,
using a repipet (Use 10 mL for clear samples, 15 mL for others).
The upper image shows the operation of the repipet.
The lower image shows the delivery of 10 mL of reaction mix to a 250 mL flask.

09_add_specimen_P5064175

2. Pipet in a precisely measured aliquot of sample specimen to be titrated into the 250 mL flask. (See separate protocol for specific volumes. The total vitamin C should not exceed 15 mg/flask).

III. TITRATE THE SAMPLE:

10_start_meniscus_P5020008

1. Follow the format for recording your data as demonstrated : Record the starting volume in the buret to nearest hundredth of a mL (remember that the graduations go from top to bottom of buret, the numbers increases as you go down, and that you read at the bottom of the meniscus). Before you begin each titration, judge whether you have enough titrant in the buret to finish the flask. When in doubt, refill the buret, and record the new start reading.

2. Place prepared flask of sample under spout.

11_add_iodine_P5064177

3. If you have not mastered it yet, practice again controlling the stopcock so that a single drop is added to the flask.

12_close_to_endpoint_P5064179
4. Add titrant while simultaneously swirling the flask. Be cautious on the first flask, as it may take much less titrant than you anticipate.

13_endpoint_close_P5064178
5. When the color change begins to show while swirling, reduce the rate of titrant addition. Continue to reduce the rate of iodine addition as you approach the endpoint (color will take longer to disappear).

6. Begin adding titrant drop-wise, swirling to remove color after each addition.
comparing endpoints

14_endpoint_P5064181

7. Stop when a trace of blue is stable (hopefully after a single drop has been added). The flask at the left displays a good endpoint. The flask in the middle has not been titrated, and for the flask at the right, the endpoint was over shot (too dark a blue).

15_finish_meniscus_P5020011
8. Record the finish buret reading to nearest hundredth mL.

9. Repeat this process for the second and third flask, using the finish reading of the previous flask for the start of the current flask.

10. The second and third flasks can be titrated more quickly since you can estimate where the endpoint will be. Shoot in titrant at full speed until about three mL short of predicted endpoint. Reduce speed markedly and finish carefully as in steps 5 through 7.
IV. CALCULATE THE RESULTS:

1. Determine the mL titrant used for each flask, determine the mean and mean deviation for the three. (If the deviation is greater than 3%, consider repeating the titration.)

2. Calculate the mean mg of vitamin C in aliquot titrated by multiplying the mean mL titrant required times the conversion factor for the iodine titrant used.

3. Calculate the mg vitamin C/100 mL sample by multiplying the mg vitamin C/aliquot X 100 mL/aliquot volume. If sample is a slurry (of vegetable, etc), further multiply mg vitamin C/100 mL times the inverse of fraction of slurry which is the starting material (i.e., if 1 g per 5 mL total, multiply times 5) for the mg vitamin C per 100 g of starting material