This document provides a standard operating procedure for analyzing the alpha and beta acid contents of hops using UV spectrophotometry. It describes preparing samples by grinding hops and extracting acids using toluene. Samples are then prepared using an auto-diluter and absorbance is measured on a UV spectrophotometer. Calculations are performed to determine alpha and beta acid contents and the hop storage index. Safety precautions are outlined for handling chemicals like toluene and methanol.

1. Page 1
Standard Operating Procedure (SOP) for UV
Spectrophotometer Analysis for Alpha and Beta Acid
Contents of Raw Hops and Hop Pellets, and Hop
Storage Index (HSI) Calculations
Created by Justin Alexander Date Created 1/21/2016
Revision # 01252016.1 Revision Date 1/25/2016
Replaces Version 01222016.1 Date Approved 1/25/2016
Reviser Justin Alexander Revision Approval Justin Alexander
1. PURPOSE & SCOPE
1.1. This method is used for the analysis of α- and β-acid contents contained within a hop sample. This method
applies to both raw hop and pelletized hop samples that have been dried and ground. These analyses will allow
for the determination of α- and β-acid contents, as well as the HSI of the sample(s) to be analyzed.
2. ENVIRONMENTAL HEALTH & SAFETY
2.1. Toluene and methanol are highly flammable. Sodium hydroxide, which is also contained within alkaline
methanol, is caustic and contact should be avoided. Promptly rinse any chemical that gets on skin. Chemical
waste should be disposed of in the proper hazardous waste containers. Further information on these chemicals
can be found in the MSDS. All chemical handling should be done with gloves and safety glasses. Be sure to
observe the expiration date on all chemicals being used.
3. CALIBRATION
3.1. The MicroLab 500 Series UV Spectrophotometer does not require calibration. The program has the necessary
equations entered so as to display the correct values for absorbance.
4. EQUIPMENT & CHEMICAL PREPARATION
4.1. MicroLab 500 Series auto-diluter with 10mL and 25µL syringes.
4.2. UV spectrophotometer with quartz cuvettes.
4.3. Analytical Scale with adequate accuracy to measure out 50 grams of sample.
4.4. Grinder/Blender.
4.5. Mechanical shaker.
4.6. 60mL sample jar with lid.
4.7. Waste beaker.
4.8. 3 Test tubes.
4.9. 20µL of Toluene.
4.10. Methanol.
4.11. 100mL of 1.2N Sodium Hydroxide (5.1).
4.12. 100mL of Alkaline Methanol (5.2).
4.13. 50 grams per sample of raw, dried hops or pelletized hops at room temperature.
5. SETUP
5.1. 1.2N Sodium Hydroxide
5.1.1. Add 50mL of DI water to a 100mL volumetric flask.
5.1.2. Adjust for purity by dividing the purity of the NaOH by 4.8 grams. Weigh out the appropriate
amount of reagent NaOH in a plastic weigh boat.
5.1.3. Add NaOH to flask and swirl to dissolve, let cool to room temperature.
5.1.4. Bring flask to 100mL total with DI water. In order to mix properly, stopper the flask and invert a
total of 10 times.

2. Page 2
5.1.5. The remaining 1.2N NaOH can be saved and used over the period of 1 month. A new batch
should be made every month, regardless of the amount remaining from the previous month.
5.2. Alkaline Methanol
5.2.1. For each 100mL needed, use 1mL of 1.2N NaOH.
5.2.2. Fill a 100mL volumetric flask approximately 50% full with methanol. Add 1mL of 1.2N NaOH, and
fill the remaining volume with methanol to achieve 100mL total.
5.2.3. Stopper the flask and invert 10 times in order to mix the contents thoroughly.
5.2.4. Decant contents of the 100mL flask to a 500mL amber glass bottle for storage.
5.3. Diluter Preparation and Shutdown
5.3.1. Turn the power on.
5.3.2. Put tubing into 500mL amber glass bottle containing the alkaline methanol.
5.3.3. Set “Size” on the left display to 10000µL. The illuminated dot will indicate 10mL. This is still
correct.
5.3.4. Set “Size” on the right display to 25µL.
5.3.5. Set “Volume” on the left display to 9980µL.
5.3.6. Set “Volume” on right display to 20µL.
5.3.7. Set “Speed” on the left display to 8.
5.3.8. Set “Speed” on the right display to 6.
5.3.9. Prime machine by pressing down the prime switch. Catch any waste methanol into a
toluene/methanol waste beaker.
5.3.9.1. Keep the tubing out of alkaline methanol solution for half of the first prime cycle.
5.3.9.2. Prime with tube completely in the alkaline methanol solution two more times or until
there are no more bubbles.
5.3.10. Prepare 2 test tubes of alkaline methanol and label them as “MeOH Blank”.
5.3.11. Prepare 1 test tube of alkaline methanol and 20µL of toluene. This process is the exact same as a
sample preparation. Fill the tube twice for 20mL total. Cover the test tube with a clean, gloved
thumb and invert twice to mix. Label this as the “Toluene Blank”. You can obtain fresh toluene
from the supplies.
5.3.12. Be sure to power off the diluter after all samples have been prepared.
5.4. Spectrophotometer
5.4.1. Allow the spectrophotometer to warm up for 30 minutes prior to being used. This ensures that
the lamp is at the proper temperature.
5.4.2. Press “F4”. This will enable PC control.
5.4.3. Open the template “Brewing Values Method” from the computer desktop.
5.4.4. Save to L:DataBrewingValueData in the appropriate folder with the file name generated by the
software.
5.4.5. Press the button that says “Connect to PC”.
5.4.6. Verify the solvents against a water sample.
5.4.6.1. Baseline with ambient air before the water sample.
5.4.6.2. This step measures the purity of the solvents used against water. Place the DI water in
the back measuring position for these steps.
5.4.6.3. Water: Baseline from 270-360nm with DI water in each measuring cell. Enter “Water” in
the sample list and select “Read Unknown”. Press “OK” to read the second sample.
Insert the cuvette in the spectrophotometer with Q always on the left.
5.4.6.4. Methanol: Rinse front cuvette with MeOH rinse bottle and then fill the cuvette from the
test tube labeled “MeOH Blank”.
5.4.6.5. Enter “MeOH Blank” in the sample list and “Read Unknown”.

3. Page 3
5.4.6.6. Dump and refill the front cuvette with the second “MeOH Blank” and “Read Unknown”.
The absorbance at 275nm must be less than 0.060. If this does not hold true, do not
proceed. Notify instructor immediately.
5.4.6.7. Toluene Methanol: Rinse front cuvette with MeOH rinse bottle and refill with solution
from the test tube labeled “Toluene Blank”.
5.4.6.8. Enter “Toluene Blank” in the sample list and “Read Unknown”.
5.4.6.9. Dump and refill the front cuvette with the “Toluene Blank” solution (be sure to save at
least 5mL for later use). Absorbance at 275nm must be less than 0.160. If this does not
hold true, do not proceed. Notify instructor immediately.
5.4.7. Dump the water contained in the back cuvette, rinse it thoroughly with the MeOH rinse bottle,
invert on Kimtech KimWipe for 20 seconds or until dry. Once dry, fill the cuvette with the
remaining 5mL of the “Toluene Blank” solution.
5.4.8. Before running the baseline, make sure that the reading is ±0.003 at 275nm. The same “Toluene
Blank” should now be in both positions in the spectrophotometer. “Baseline” from 270-360nm.
5.4.9. Enter “Blank” in the sample list and “Read Unknown”. Press “OK” and read the sample a second
time. The blank should read ±0.003 across all absorbance wavelengths. If it is outside of the
appropriate range, return to step 5.4.6.
5.4.10. You are now ready to run all samples.
5.4.11. Be sure to power off the spectrophotometer after all samples have been analyzed.
6. PROCEDURES
6.1. Allow hops to reach room temperature.
6.2. For raw or pelletized hops, grind at least 50 grams, homogenize in a mixing bowl, and refill the sample jar with
the ground hops until full.
6.3. Label a 60mL toluene extract bottle with the sample ID.
6.4. Place 2.500 grams (±0.002) of ground hops in a sample jar and seal the lid, taking care to avoid adding chunks.
6.5. Add 50.0mL of toluene to all bottles containing ground hops and reseal the lid. This measurement is essential
to the proper function of the instrument. Measure out carefully and accurately.
6.6. Move sample jars to shaker and shake on high for 30 minutes.
6.7. Allow samples to rest for 5 minutes, allowing the hop material to separate.
6.8. Cycle the auto-diluter once with the hop sample, making sure that the dispensing liquid is moved into the
waste container. This will help clear out any air bubbles that have formed in the auto-diluter.
6.9. Rinse the sample cuvette with methanol inside and out and allow it to drain upside down on a Kimtech
KimWipe for at least 20 seconds or until dry.
6.10. Using the auto-diluter, aliquot 2 samples into clean test tubes. Mix sample thoroughly using a vortex, if
possible. The auto-diluter will mix 9980µL of alkaline methanol with 20µL of the sample. Avoiding air bubbles
during this step is essential for an accurate reading.
6.11. Rinse cuvette from the first aliquot with methanol, allow to dry upside down on a KimWipe for 20 seconds,
and then fill the cuvette from the same test tube. It is essential that the cuvette is properly cleaned and free of
blemishes.
6.12. Place the cuvette in the UV spectrophotometer.
6.13. Enter the sample ID in the sample list and “Read Unknown”.
6.14. Once the reading has finished, empty the cuvette into a waste beaker, rinse with a second aliquot, and then fill
from the same test tube.
6.15. Press “OK” to read the second sample.
6.16. If absorbance at 275nm differs by ±0.01 or more between the two identical samples, you must rerun the
sample. If this happens, enter the sample ID again and append “.1” to it.
6.17. Repeat steps 9-16 for remaining samples.

4. Page 4
7. RESPONSIBILITIES
7.1. α -Acid contents, β-acid contents, and HSI results can be reported for any sample upon request or as assigned.
8. FORMS & ATTACHMENTS
8.1. Refer to the MicroLab 500 Series Lab Manual for any additional maintenance. A hardcopy of the manual can be
found in the lab, or the online version can be found here. Maintenance should be performed by a qualified
technician or instructor.
9. REFERENCES
9.1. ASBC Hops Method 6A: α- and β-Acids in Hops and Hop Pellets, α- and β-Acids by Spectrophotometry.
9.2. ASBC Hops Method 12: Hop Storage Index.
9.3. MicroLab Manual: http://www.microlabtech.co.uk/documents/literature/ML500A%2069175_MNL_Rev_D.pdf.