1 Ultraviolet – Visible Spectroscopy for Determination of α- and β-acids in beer hops Introduction In this lab, you will undertake a simple extraction of two hop samples followed by spectrophotometric analysis at three wavelengths to quantify the quantities of α- and β-acids, as well as a third component linked to hop degradation. UV-Vis spectroscopy (or spectrophotometry) is one of the most important quantitative spectroscopic techniques available in the modern arsenal of analytical instrumentation for molecular analysis. The wavelength range extends from about 190 – 750 nm, which corresponds to molecular electronic transitions of different origins. Consider the electronic energy level diagram of a typical molecule (Figure 1): The four transitions correspond to: 1. σ – σ * transitions This transition type requires large energy, which may result in bond breaking. It is not very important from an analytical point of view. This type of transition typically requires energetic photons, below 190 nm. 2. π – π * transitions This is the most analytically useful type of transition. The energy required for this type of transition is moderate, corresponding to photons in the 190 – 750 nm range. 3. n – π * and n – σ * transitions Figure 1: Energy level diagram representing electronic transitions for a typical molecule
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1
Ultraviolet – Visible Spectroscopy for Determination of α- and
β-acids in beer hops
Introduction
In this lab, you will undertake a simple extraction of two hop samples followed by
spectrophotometric analysis at three wavelengths to quantify the quantities of α- and β-acids,
as well as a third component linked to hop degradation.
UV-Vis spectroscopy (or spectrophotometry) is one of the most important quantitative
spectroscopic techniques available in the modern arsenal of analytical instrumentation for
molecular analysis. The wavelength range extends from about 190 – 750 nm, which
corresponds to molecular electronic transitions of different origins. Consider the electronic
energy level diagram of a typical molecule (Figure 1):
The four transitions correspond to:
1. σ – σ* transitions
This transition type requires large energy, which may result in bond breaking. It is not very
important from an analytical point of view. This type of transition typically requires energetic
photons, below 190 nm.
2. π – π* transitions
This is the most analytically useful type of transition. The energy required for this type of
transition is moderate, corresponding to photons in the 190 – 750 nm range.
3. n – π* and n – σ* transitions
Figure 1: Energy level diagram
representing electronic transitions for
a typical molecule
2
Molecules containing lone pair(s) of electrons exhibit some special properties. Electrons that do
not participate in chemical bonds can absorb energy and are excited either to the π* (if the
molecule has π – bonds) or σ* state. Unfortunately, these transitions are also not very useful
analytically.
Spectrophotometric absorbance features in the UV-Vis tend to be quite broad. That is, the
molecule absorbs significantly over a wide range of wavelengths. For example, the absorbance
spectra of two dyes commonly used in lasers are shown in Figure 2.
Beer-Lambert Law
The analytical utility of UV-vis photon absorption derives from the (intuitive) fact that the
number of photons absorbed by an absorbing species depends on the number of absorbing
molecules and, therefore, on its concentration. The Beer-Lambert Law states that the
concentration of an absorbing species is logarithmically related to the fraction of light
transmitted (T): this is termed the “absorbance, A.” So, A = -log (T). But, you would also expect
the absorbance to depend on the “strength of the absorber,” as well as the “thickness of the
absorber.” For example, if you had a 1-mm thick piece of light absorbing plastic, you would
expect that a plastic piece that is twice as thick would absorb twice as much light. So, putting
this all together, the Beer-Lambert Law states
𝐴 = − log(𝑇) = ε𝑏𝐶
where ε is the molar absorptivity (M-1 cm-1) or “strength of the absorber”. Note that ε could
also be expressed in L/g if concentration is expressed in g/L. This is called the specific
absorptivity
b is the pathlength (cm) or “thickness of the absorber”
C is the concentration (M-1) of the absorber
Figure 2. Absorbance spectrum of Nile Blue and Coumarin 30 in
ethanol. Note that absorbance is given in arbitrary units.
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
200 300 400 500 600 700
Ab
sorb
an
ce (a
.u.)
Wavelength (nm)
Absorbance of dyes in ethanol
Nile Blue
Coumarin 30
3
The molar absorptivity is the most important indicator of analytical sensitivity in UV-Vis. When
“b” is 1-cm (most common) and A is plotted as a function of concentration, a straight line
results with a slope equal to the molar absorptivity. So, in effect, the molar absorptivity is the
change absorbance per unit change in concentration.
As with any other instrumental method of analysis, the response must be calibrated for analyte
concentration and this is the most common approach for samples that do not present any
interference with measurement of the absorbance. Owing to the broad absorption spectra
exhibited by most solvated molecules, one of the most common interferences in UV-Vis
spectroscopy comes from the presence of multiple absorbers in the same sample. For example,
is a sample contained only Nile Blue and Coumarin 30, then calibration curves could be derived
for each dye at its wavelength of maximum absorbance (without fear of the absorbance by one
of the dyes interfering with measurement of the other dye). If, on the other hand, the sample
also contained Coumarin 1 (Figure 3), then it is clear that independent calibration curves could
not be developed for the two Coumarin dyes, as their absorbance spectra overlap in the range
350 – 450 nm. It would therefore be impossible to measure independently the absorbance of
one of the dyes in the presence of the other.
Fortunately, absorbance is an additive property, and so the total absorbance (AT) and any single
wavelength is equal to the sum of the absorbance by all absorbers at that wavelength:
𝐴𝑇(𝜆1) = ∑ 𝐴𝑖(𝜆1)
𝑖
= ∑ ε𝑖(𝜆1)𝑏𝐶𝑖
𝑖
𝐴𝑇(𝜆2) = ∑ 𝐴𝑖(𝜆2)
𝑖
= ∑ ε𝑖(𝜆2)𝑏𝐶𝑖
𝑖
Figure 3. Absorbance spectrum of Nile Blue, Coumarin 30 and Coumarin 1 in ethanol. Note that absorbance is given in arbitrary units.
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
200 300 400 500 600 700
Ab
sorb
ance
(a.
u.)
Wavelength (nm)
Absorbance of dyes in ethanol
Nile Blue
Coumarin 30
Coumarin 1
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Therefore, by measuring the total absorbance at the absorption maximum of each component,
and knowing the “strength of absorption” of each component at each wavelength, we can solve
the set of equations simultaneously to determine the concentration of each absorber.
Analysis of α- and β-acids in beer hops
Hops contain both humulones (α-acids) and lupulones (β-acids), the major chemical
components of which are shown in Figure 4.
It is important for a brewer to know the quantity of α–acids in the hop used because α–acids
isomerize to form iso-α–acids during the brewing process, adding bitterness to balance the
flavor of the finished beer.
Materials
You will be provided with two samples of dried, commercial hops used for home brewing.
Additionally, you will use reagent grade NaOH and spectrophotometric grade methanol for the
acids extraction.
The spectrophotometer you will use for this experiment is the Shimadzu UV2450 double beam
instrument. Operating instructions are provided on the Chem 219 BlackBoard site.
Hazards
Methanol is highly flammable and toxic by inhalation, ingestion, or skin absorption. Waste
methanol should be placed in a labeled non-halogenated waste container for disposal (see your
TA). Sodium hydroxide (NaOH) is corrosive and can cause severe burns. Wear suitable personal
protection equipment.
Procedure: (To be conducted in triplicate)
Figure 4. Structures of major α - and β-acids found in hops.
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Prepare 250 mL of methanolic NaOH by mixing 0.5 mL of 6 M NaOH into 250 mL methanol.
Grind hop pellets approximately 1 gram at a time (otherwise you will clog the grinder) and
combine until you have approximately 8-9 g of each type of hop.
Pipette exactly 50.0 mL of methanol into a 100 mL beaker. Accurately weigh out (to the nearest
mg) 2.5 g of ground hops and add to the beaker containing the methanol. Add a stir bar, place
on a magnetic stir plate and stir for 30 min at room temperature. Allow to stand without
stirring for 10 min to let the particulate matter settle. Gravity filter the settled solution into a
separate, clean, dry 125-mL Erlenmeyer flask. [Note: Do not use additional solvent to wash the
beaker or the filtered solid (why?)]. Pipette a 50 μL aliquot of the filtrate into a 25-mL
volumetric flask and bring up to volume with the methanolic NaOH you prepared.
Fill a 1-cm quartz cuvette approximately ¾-full with blank solution (this is the methanolic NaOH
solution you used to prepare all your samples) and place it in the reference side cell holder.
Rinse a separate cuvette several times with your hops extract and finally fill the 1-cm quartz
cuvette about ¾-full with an extract solution. Place this cuvette in the sample cell holder.
Measure a background corrected absorbance spectrum of the extract solution from 210 nm to
510 nm.
Data analysis
Because there are two major components in the hop extract, the mixture of α- and β-acids
appears to be an ideal system for the classic two-component analysis covered in most
quantitative analysis texts. All that is needed is the molar absorptivity coefficients at two
different wavelengths for each component, so a system of two equations can be solved for two
unknowns. The “fly in the ointment, or beer in this case” is that the hops natural product
extract is more complex. Firstly, neither the α-acids nor the β-acids are single compounds (see
Fig. 4). Additionally, there is a third component that appears over time as the α- and β- acids
are degraded. This third component has not been purified and is thought to be some other
breakdown component of the hops. It absorbs strongly at 275 nm but has significant
absorptions at 325 and 355 nm, where it augments the absorption of α- and β-acids and
interferes with a standard two-component analysis.
Because we are not dealing with single absorbing compounds, it is more practical to use specific
absorptivity than molar absorptivity. Specific absorptivity relates the absorbance measurement
to that of a mixture of compounds with a total concentration of 1 g/L at a given path length
(typically 1 cm). Alderton et al. (Alderton, Bailey et al. 1954) reported specific absorptivities (L g-
1 cm-1) for pure α- and β-acids, as well as the third degradation component. These are given in
Table 1.
355 nm 325 nm 275 nm
α-acids 31.8 38.1 9.0
6
β-acids 46.0 33.1 3.7
Comp3 1.0 1.5 3.1
Table 1: Specific absorptivities (L g-1 cm-1) for α- and β-acids in beer hops, as well as the third
absorbing component (see Ref. 1).
Because the total absorbance at any wavelength is equal to the sum of absorbances by each
component, we can describe this three-component system with three equations:
𝐴355 = 31.8𝐶𝛼 + 46.0𝐶𝛽 + 1.0𝐶𝐶𝑜𝑚𝑝3
𝐴325 = 38.1𝐶𝛼 + 33.1𝐶𝛽 + 1.5𝐶𝐶𝑜𝑚𝑝3
𝐴275 = 9.0𝐶𝛼 + 3.7𝐶𝛽 + 3.1𝐶𝐶𝑜𝑚𝑝3
Although this system of three equations can be solved by any graphing calculator (no duh), we
will use the method of Gaussian elimination (which is what your calculator is doing under the
hood). The method of Gaussian elimination can be used to solve virtually any system containing
an equal number of unknowns and equations. An introduction to the Gaussian elimination
method is given in the Appendix.
Of course, the brewer is not interested in knowing the concentration of α- and β-acids in an
extract, but rather wants to know the percentage of α- and β-acids in the actual dried hop. So,
your final task is to work through your procedure and dilutions to find this number. Be sure to
report appropriate statistics for your results.
Analysis point 1. From the overall shape of your absorbance curve, decide if your sample contains mostly humulones (alpha-acids) or lupulones (beta acids).
Analysis point 2. Make a data table containing the absorbance values for all solutions at 355 nm, 325 nm, and 275 nm. Use the data to do a two component analysis of your spectra and determine the grams of humulones and lupulones in your extract and the % alpha and beta acids in your original hops sample.
Analysis point 3. Use a three-component analysis to find the concentration of α- and β- acids in the solutions you analyzed. How do these values compare to your two-component analysis? Next, use your values from the three component analysis to determine the concentration of α- and β- acids in the actual extract. Finally, determine the % α- and β- acids in the original hops sample. How do they compare to your two-component analysis?
Alderton, G., et al. (1954). "Spectrophotometric Determination of Humulone Complex and Lupulone in
Hops." Anal. Chem. 26: 983 - 992.
Appendix: Mathematical Background using the Hops Data as a Real World Example
7
The hops lab provides an opportunity to solve a system of linear equations with algebra and
matrices. Here you can practice methods using real-world numbers rather than the artificial
whole numbers that are typically used in mathematics texts, designed to work without fractions
or decimals.
The absorbance of a hops solution at three key wavelengths can be found (assuming a 1 cm path