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Do you know what's in your beer?

Citrus, grapefruit, toffee, spice, apple, pear, biscuit, caramel… the list of flavors and aromas associated with beer is vast but how are they produced during the brewing process?  The hop variety used in the brewing process is responsible for the bitterness, aroma, and flavor characteristics.  These compounds can be extracted from hops by steam distillation, liquid-liquid extraction and analyzed using GC-MS.
John Langley and Julie Herniman (University of Southampton) run an undergraduate laboratory experiment extracting aroma compounds from hops. This advanced practical was designed to extend practical knowledge and experience, particularly in the field of analytical chemistry.  The students are required to deliver laboratory notes, a 'Report for the Chief Brewer' and a poster. Their work is assessed for technical understanding, and uncertainties and errors associated with the whole practical and processing of their
GC-MS data.

The students undertake the extraction, sample submission and analysis, and have access to CHROMacademy to help them answer specific and general questions posed during the experiment.

The history of beer making goes back over 6000 years, whilst hops were introduced into this process around 1200 years ago.  Malt was first used in the middle 17th century and the beers we recognize today are a product of the Free Mash Tun Act (1880) where the tax on malt was replaced by a tax on sugar. The new law then enabled "the brewer to brew from what he pleases and have a perfect choice of his materials and methods".

There are 20 commercially grown British Aroma Hop varieties in the UK.  The British Hop aromas include notes such as tangerine, citrus, grass, grapefruit, chocolate, blackcurrant, spice, pepper, apricot, marmalade, and mint.1  Hops are the female flowers (also called seed cones or strobiles) of the hop plant, humulus lupulus (Figure 1).  A hop cone consists of layers of soft tissue petals, within which lupulin glands form (these look like large yellow pollen grains).  It is the lupulin glands that contain the oil resins which provide the bitterness, preservative properties, flavor, and aromas to beer.


Figure 1: Hops
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Understanding the chemical composition of the vast varieties of the different hops is an important part of the brewer's art.  Variation in the relative amounts of alpha acids and volatile oils impart specific flavors to the final product, e.g. citrus/grapefruit often found in American hops.1  Most brewers will use a combination of a variety of different hops which can also be added at different times during the brewing process, in order to exploit certain flavor and aroma features.

The two major types of chemical compound from hops which provide the bittering, flavor, and aroma characteristics to beer are acids and essential oils.2  The acids contained within hops are alpha acids, humulone, adhumulone, and cohumulone, (Figure 2) which form iso-alpha acids when the hops are boiled during the brewing process, and beta acids, lupulone, colupulone, and adlupulone (Figure 3). 

Alpha acids found in hops

Figure 2: Alpha acids found in hops.

: Beta acids found in hops

Figure 3: Beta acids found in hops.


Each hop variety contains a different percentage of alpha acid; the total alpha acid value is reported to allow the brewer to calculate the desired bitterness of the beer.  During the boiling process in brewing the less soluble alpha acids are isomerized to their more soluble corresponding iso-alpha acids which impart the primary bittering to the beer (Figure 4).3  This is why bittering hop additions are normally done at the beginning of a boil of 60 minutes or longer.


Humulone isomerization to the corresponding iso-alpha acid isohumulone

Figure 4: Humulone isomerization
to the corresponding iso-alpha
acid isohumulone.3


Beta acids are rarely considered separately, however, they are also important in giving flavor to beer.  They do not produce as much bitterness as alpha acids during the boiling process, however, they do create bitterness through oxidation during fermentation and storage, thus, they can affect the long-term character of a beer.  The beta acid content of hops is given either as a total percentage of beta acid in the hops by weight or as a ratio of alpha to beta acids.

The essential oils found in hops are primarily humulene, myrcene, caryophyllene, and farnesene (Figure 5).  These compounds contribute most of the flavor and aroma to beer.  The total percentage of essential oils in a hop is given by weight and sometimes the identity of a specific oil will be given as a percentage of the total oil.  Hop essential oils are volatile, therefore, in traditional brewing processes the flavor characteristics of hops were obtained by late addition of hops or dry hopping, which allows the flavor compounds to be absorbed and not boiled off.   As dry hopping does not involve any heat it does not extract any bitterness from the hops; it is solely for the purpose of adding more hop aromas and flavors to the beer.  The addition of the hops for flavor was often undertaken in the last 15 minutes or less of the boil, while aroma hops will only be added when there is 5 minutes or less of boiling remaining.  Recently, brewers have experimented with different addition times of the hops to produce beer with different flavor profiles.

Essential oils found in hops

Figure 5: Essential oils found in hops.


To typify a hop variety or determine the freshness of the hop the volatile oils can be extracted and then analyzed by gas chromatography-mass spectrometry (GC-MS).  This can be done using a steam distillation process, followed by a liquid-liquid extraction, and sample concentration before final analysis by GC-MS.  From the resultant chromatogram and electron ionization mass spectra the variety of hop should be identifiable.

Beer can be produced on any scale from small home brews, to craft micro-breweries, and finally large industrial scale.  However, the brewing process is ultimately very similar on each scale (Figure 6).  Although, as has been mentioned previously, each individual brewer will alter the time at which the hops are introduced in order to exploit certain bittering and aroma characteristics.

Brewing process

Figure 6: Brewing process

Hop Analysis Method    

Equipment and Experimental


2 x 2-neck 250 mL round bottom flask
1 x 1-neck 250 mL round bottom flask
1 x 250 mL separating funnel
1 x heating mantle
1 x water cooled condenser
1 x Claisen head (distillation head)
1 x vented side arm
1 x 150 ˚C alcohol thermometer

2 x stands with bosses & clamps
1 x 250 mL beaker
1 x 25 mL beaker
2 x 5 mL volumetric flask
1 x 50 mL measuring cylinder
1 x large plastic funnel
GC-MS vials & lids
Pasteur pipettes

Weigh out approximately 15 g of hops and add to a 250 mL two-necked round bottom flask using the plastic funnel together with 130 mL of de-ionized water.  Place this vessel in a heating mantle and construct the distillation apparatus as shown in Figure 7.  Add 250 mL of de-ionized water into the dropping funnel. Heat the aqueous hop mixture, reflux for 2 hours and record the reflux temperature. Carefully add de-ionized water to the heated vessel during the distillation such that the volume of distillate collected equates to the volume of water added.  Note, do not add the water too quickly.  Record the volume of distillate and the volume of water added via the dropping funnel.
Distillation apparatus

Extract the distillate with one aliquot of diethylether (50 mL) using a separating funnel. Transfer the ether layer into a clean beaker and remove the majority of the solvent using a water bath until 1‑2 mL of the extract solution remains.  Transfer this to a 5 mL volumetric flask and make up to 5mL with diethylether.

Transfer ~1mL of the final solution of the hop extract to a GC vial using a Pasteur pipette and submit this for GC-MS analysis.  Two GC-MS methods are used for analysis; a split and a splitless method.  Both of these methods can be useful in analyzing the compounds extracted from the hops.  The splitless method (which allows the whole sample to be transferred to the column) can allow for the identification of low concentration components extracted from the hops.  Although these components are only present in very low concentrations they may be important in contributing to the final beer product.

The instrument settings and the resultant chromatograms for two hop varieties (Galena and Challenger) are shown in Figure 8.  It can be seen that the chromatograms, from student experiments, are highly complex yet distinct for the two hops varieties allowing for identification of each.

Figure 7: Distillation apparatus.

Galena and Challenger hops
Oven Method  
Initial Temperature (°C):  40
Initial Time (min):   4.50
Number of Ramps:  1
Rate #1 (deg/min):   20.0
Final Temperature #1 (°C): 320
Hold Time #1 (min):   7.00
Post Run Temperature: Off
Enable Cryogenics:  Off
Maximum Temperature (°C): 350
Prep Run Timeout (min):  10.00
Equilibration Time (min):   0.10
Right SSL Method  
Base Temperature:  On
Base Temperature (°C):  220
Mode:  Splitless
Split Flow:   On
Split Flow Flow (mL/min): 60
Splitless Time (min):  1.00
Surge Pressure:  Off
Surge Pressure (kPa) :  3.00
Surge Duration (min): 0.00
Constant Purge: On
Stop Purge At: (min): 0.00
Right Carrier Method  
Mode:   Constant Flow
Initial Value:  On
Initial Value (mL/min):  1.20
Initial Time:  1.00
Gas Saver:   On
Gas Saver Flow (mL/min):  20
Gas Saver Time: 5.00
Vacuum Compensation:  On

Figure 8: Analysis conditions
and chromatograms for Galena
and Challenger hops.


As has been mentioned previously, hop growers will analyze and report the percentage of acids and oils in a hop variety in order to allow the brewer to produce a beer with the desired characteristics of bitterness, flavor, and aroma.  Table 1 details the chemical composition of Challenger and Galena hops.

Hop Variety Challenger Galena
Alpha acid (%) 6.5-9.0 12.0-14.0
Beta acid (%) 3.2-4.2 7.0-9.0
Co-humulone (%) 20-25 37-42
Total Oils 1.0-1.5% 0.9-1.2 mL/100 g
Myrcene (%) 30 55-60%
Humulene (%) 25 10-13
Caryophyllene (%) 8 4-6
Farnesene (%) 1 < 1
Aroma Spicy, cedar, green tea Spicy, blackcurrant and citrus (grapefruit)

Table 1: Analysis of challenger and galena hops.4-6


It has been shown that relatively simple experimental procedures can be used to extract, analyze, and differentiate between different hop varieties.  The process of combining these different hop varieties, which is common practice in brewing, to produce a unique beer is a much more subtle art. 



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  2. //; //
  3. De Keukeleire, D.; Verzele, M. Tetrahedron 1971, 27, 4939-4945.
  4. //
  5. //
  6. //
John Langley

John Langley

John is Head of Characterization and Analytics, at the University of Southampton.  He has nearly 30 years' experience of MS and has been responsible for the MS Facility in Chemistry at the University of Southampton since 1988. His interests are the application of mass spectrometry as a routine tool for chemistry/chemical biology/oilfield chemistry as well as probing new areas for research using MS and separation science-MS. Specific interests are the application of  hyphenated approaches (GC-MS, HPLC-MS & SFC-MS) to analysis of petrochemicals, particularly biofuels, research into analysis and detection of oligonucleotides and understanding fundamental MS/MS fragmentation processes and mechanism, the latter including the use of computational methods.

John is a Chartered Chemist, Chartered Scientist and a Fellow of the Royal Society of Chemistry (RSC) and an IUPAC Fellow; he is Chair of the RSC Separation Science Group and Secretary of the International MS Foundation; member of the Editorial Board of Rapid Communications in Mass Spectrometry; formerly Chair of the British MS Society (2008-2010), Vice-Chair (2006-08) and a member of the BMSS advisory Board (2012-2017). He was the European representative of the IUPAC working group for Standard Definitions of Terms Relating to MS and is a member of the EPSRC Peer Review College.


Essential oils found in hops


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