Flavor Analysis Laboratory

 

For this laboratory, we will use five different methods to extract the volatile (aroma) compounds from an aqueous extract. You will be provided with about 100 ml of a 1 ppm solution of 10 aroma compounds in water. You need to apply the method of volatile isolation you have been assigned to isolate these volatiles in a manner such that they can be analyzed by gas chromatography. Gas chromatographic operating parameters will be provided in lab. Abbreviated instructions to perform each isolation procedure follow below - detailed instructions will be provided in laboratory..

We will pool at the class data and ask you for a discussion of the results in place of an internet assignment.  We will be looking for an explanation as to why we find the results we do (within the 2 page assignment limit).  

 

1.  Likens-Nickerson Simultaneous Distillation and Extraction

Sample (100 ml) and extraction solvent (dichloromethane) are placed in separate flasks.  Both flasks are heated to their respective boiling point temperature) and the solvent vapor (reasonably pure) and the sample vapor mix and re-condense in the collector. Because of their density differences, the two liquid phases separate and return to their respective flasks. The product steam strips flavor components from the food and the solvent extracts the flavor compounds from the condensed steam. After 1 hr extraction,  the run is stopped. The solvent extract is then dried with about 3 g of anhydrous MgCl2, filtered (after sitting 5 min) and concentrated to 0.5 ml. This sample is suitable to inject into a gas chromatograph. 

The 100 ml sample and extraction solvent, dichloromethane, were placed in separate flasks.  After assembling the apparatus, the system was primed by adding some dichloromethane to the collector vessel, which gives the condensate a place to separate before flowing back to their respective
flasks.    Both flasks were heated to their respective boiling point temperature and the solvent vapor (reasonably pure) and the sample vapor
mixed and re-condensed in the collector. Because of their density differences, the two liquid phases separated and returned to their
respective flasks. The product steam striped flavor components from the food and the solvent extracted the flavor compounds from the condensed
steam. After the 1 hr extraction, the run was stopped. The solvent extract was then dried with about 3 g of anhydrous MgCl2, filtered (after sitting 5
min) and concentrated to 0.5 ml. This sample was injected into the gas chromatograph

2.  Static headspace analysis

For this extraction, one takes a certain volume of air above a food and injects it into a GC. We make sure that the sample has adequate time for volatiles in the food to equilibrate with the air. This method can be performed in several ways and may be a manual or automated process. These variations will be discussed in the lecture portion of the class.

We will be using a manual process. Place about 5 ml of aqueous sample into a 20 ml headspace vial and allow the vial to sit for about 30 min at 50C. Withdraw 2 ml of headspace and inject it into a gas chromatograph. Make a very fast injection. Repeat the whole process using a new sample but make a slow injection (1.0 ml/min).

This extraction, one takes a certain volume of air above a food and injects it into a GC. We make sure that the sample has adequate time for volatiles in the food to equilibrate with the air. This method can be performed in several ways and may be a manual or automated process. These variations will be discussed in the lecture portion of the class.

We did the process manually. We started the by obtaining two 20 ml headspace vials, 2 silicone septa and 2 metal caps.  Then place about 5 ml of aqueous sample into each 20 ml headspace vial and close with the lids. Next, we set up 2 sets of heating water bath by using two hot plates, clamps, temperature probes, and beakers with water. After that we allowed the vial to sit in the hot water at 50C. The first vial sat in the hot bath for about 30 min and the second vial sat in the hot bath for about 1 hour and 15 minutes. The second vial sat in longer because we were waiting for the GC to complete the first run. Note also that only the half of the bottom half of the vial was in the hot water and not the whole vial.

GC procedure: we withdrawn 2 ml of headspace and injected it into a GC. We ran the first sample with a very fast injection (15 seconds injection) and second sample with a slow injection (2 minutes injection). Note again that before each injection, we cooled the initial part of the loop with liquid nitrogen in order to cryofocus. The cryofocus was done because 2 ml of the sample was a large amount. If we did not do this, the whole sample would not get through the columns at the same time, and thus the GC would give several sets of same compound detection.

 

3.  Solvent extraction

We will be doing a simple solvent extraction (dichloromethane and hexane) using separatory funnels. Place 100 ml of the sample into two 250 ml separatory funnels.  Extract one 3 times with dichloromethane and the other 3 times with hexane. Pool the extracts and dry them with about 3 g of anhydrous MgCl2. Evaporate the solvent extracts to about 0.5 ml and inject the sample into a gas chromatograph.

100mL of sample was placed into each of two 250mL separatory funnels.  (I left the actual amounts of sample at home today, but they were around 95mL each.)  Samples were extracted with solvent (dicloromethane and PENTANE, a change from the handout) by adding 50mL solvent to each flask, inverting the flask 50 times, and collecting the solvent phase.  This was performed three times on each funnel, and the solvent phases of each funnel were pooled.  The two resulting extracts were evaporated by nitrogen flushing to a final volume of 0.5mL and analyzed by GC by our gracious TA, Dana.   During the extractions, the aqueous phase of the dicloromethane extraction appeared cloudy while the pentane extraction had a clear aqueous phase.  Also, the pentane extraction was performed in a slightly leaky seperatory funnel, which could lead to decreased recovery amounts.

4.  Solid Phase Micro Extraction (SPME)

This process is essentially a combination headspace and absorption methods.  The SPME fiber is coated with an absorbent which will "extract" volatiles from the sample headspace. (Several different absorbents are available) The absorbed volatiles are desorbed in the injection port of a gas chromatograph.

Three Solid Phase Micro Extraction (SPME) fibers were employed in this laboratory for analyzing the 13 flavors present in the provided 10 ppm solution.  Specifically, the SPME fibers used in this laboratory were 75 mm Carboxen/polydimethylsiloxane (black), 85 mm  polyacrylate (white), and 65 mm polydimethylsiloxane/divinylbenzene (blue).  The procedure for extracting volatiles from the solution was the same for each fiber, and is outlined below.

SPME Procedure 

  1. Extracting volatiles
    1. Put 5 mL of solution into a headspace vile.
    2. Cap the vile with Teflon-lined septum and crimp shut.
    3. Insert SPME syringe through the septum and then push down SPME fiber from within the syringe, exposing as much fiber as possible without contacting it with the solution or the sides of the vile.  To ensure that the fiber would not touch anything while in the vile, the syringe holder was held by a clamp on a rod and the vile by a wire rack.
    4. Let the fiber stand in the headspace for 30 minutes, then retract fiber into syringe again and pull syringe out of the vile.
  2. GC Program
    1. With the oven off and cooled, unwind the first few loops of the GC column and put into flask of liquid nitrogen. 
    2. Insert syringe into GC injector (220C), then lower SPME fiber. 
    3. Keep SPME fiber in the GC injection port for two minutes, causing desorption of volatiles from the fiber during this period of cryogenic focusing.
    4. After the two minutes has passed, take the column out of the liquid nitrogen and return it to it original position in the oven. 
    5. Turn the oven on and let the GC program run:

                                                                          i.      2 minutes at 45C
ii..      Ramp temperature up at a rate of 10C per minute until oven temperature reaches175C.
iii.       Hold oven temperature at 275C for 10 minutes.

    1. Remove SPME fiber and syringe from GC injection port 13 minutes after starting the GC program.  (Leaving the fiber in the port for some time after cryogenic focusing ensures that all volatiles are desorbed from fiber.)

*Note:  The 85 mm  polyacrylate (white) SPME fiber was accidentally left in the injection port for 15 minutes after starting the GC program instead of only 13 minutes. 

 

5.  Solvent Assisted Flavor Extraction (SAFE)

This method can be directly applied to a food (aqueous base) or to the solvent extraction of a food product. The method consists of dripping the food (or food extract) into a high vacuum area where the solvent flash evaporates carrying the volatiles in the food with it. The volatiles and solvent are condensed and collected. They are dried to remove water, concentrated and analyzed by GC. Details on the method will be given in the laboratory.

 This method can be directly applied to a food (aqueous base) or to the solvent extraction of a food product.  The 100 ml of aroma compounds and 50 ml of methylene chloride were mixed and added. The method consists of dripping the food (or food extract) into a high vacuum area where the solvent flash evaporates carrying the volatiles in the food with it. A small amount of sample is added to the vacuum area at a time because too much will cause the vacuum to be lost.  The water bath and jacket temperature were kept at 38C.  The vacuum had a range of 1X10-4 to 6X10-5 Torr.  The volatiles and solvent are condensed and collected. Liquid nitrogen is used to cool the sample.  The whole vapor flask is covered in liquid nitrogen.  It took 20 minutes to drip the sample in and it sat for about a half hour.  It was not completely done when it was removed.  It could have sat for another two hours to get more of the volatiles out.  The pump was shut off and the vacuum was released slowly.  The sample was thawed and the methylene chloride and water were separated using a separatory funnel.  The methylene chloride was removed from the sample using a hose to evaporate the solvent.  They are dried to remove water, concentrated and analyzed by GC. Details on the method will be given in the laboratory.

Aroma Compounds Listed in Order of Elution from a DB-5 column

 

Aroma

           Compounds

    DB-5 LRI

1

Fresh Yogurt

Acetaldehyde

<500

2

Butter

Diacetyl

0612

3

Strawberry

Ethyl butyrate

0799-0800

4

Almond/Cherry

Benzaldehyde

0924-0968

5

Roasted

2-Acetylpyrazine

1023

6

Orange

Limonene

1031-1041

7

Strawberry

Maltol

1111

8

Lemon

Citral

1254

9

Clove

Eugenol

1356-1364

10

Vanilla

Vanillin

1391-1410

11

Cinnamon

trans-Cinnamaldehyde

1467

12

Peach

γ-Dodecalactone

1473

LRI values obtained from:
http://www.nysaes.cornell.edu/flavornet/chem.html
http://www.odour.org.uk/