CHAPTER TWENTY-THREE

Reverse-phase HPLC Analysis andPurification of Small MoleculesKirstie Canene-Adams1Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD, USA1Corresponding author: e-mail address: kirstieadams26@yahoo.com

Contents

1.

MetISShttp

Theory

hods in Enzymology, Volume 533 # 2013 Elsevier Inc.N 0076-6879 All rights reserved.://dx.doi.org/10.1016/B978-0-12-420067-8.00023-4

292

2. Equipment 293 3. Materials 294

3.1

Solutions & buffers 294 4. Protocol 295

4.1

Preparation 295 4.2 Duration 295

5.

Step 1 Break Down the Matrix of the Samples that Contain the Molecule of Interest 295 5.1 Overview 295 5.2 Duration 296 5.3 Tip 296 5.4 Tip 297 5.5 Tip 297 5.6 Tip 297 5.7 Tip 297

6.

Step 2 Extraction of Carotenoids from the Samples 297 6.1 Overview 297 6.2 Duration 297 6.3 Caution 298 6.4 Tip 298

7.

Step 3 Carotenoid Analysis on a Reverse-Phase HPLC-PDA System 299 7.1 Overview 299 7.2 Duration 299 7.3 Tip 300 7.4 Tip 300

References

300 Source References 301

Abstract

Reversed phase high-performance liquid chromatography (HPLC) is utilized for the sep-aration of molecules due to their polarity in order to quantify, identify, and/or purifyvarious samples such as those from serum, human and animal tissues, drugs, and foods.

291

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292 Kirstie Canene-Adams

The following protocols are for extracting carotenoids from samples and subsequentHPLC analysis. If you are interested in another compound for HPLC analysis, Sigma-Aldrich® has a wonderful online resource for multiple adaptations to the HPLC protocolfor the analysis of hundreds of compounds (see References).

1. THEORY

Molecules from a sample, which can be manually injected one at a

time into the machine, or automatically injected with computerized sample

loading injectors, bind to a reverse phase HPLC column when first an aque-

ous, polar mobile phase (e.g., buffered water) is pumped through the system.

The sample is eluted off the column with an organic mobile phase that is less

polar (e.g., acetonitrile), Fig. 23.1. The samples can be collected as they

come off the column for the purpose of purification. Most HPLC systems

have two pumps in order to gradually mix these two solvents, or mobile

phases, as they are more commonly called. HPLC columns vary greatly

and come in two categories: analytical or preparative, the major difference

being that the preparative columns are bigger so they can accommodate

ure 23.1 Diagram of HPLC System. Arrows show the flow of mobile phases, of whichre are two, A and B, which are mixed in the dual pump system. A sample enters theem via either a single manual injection or using an automated injection system.re are various types of columns depending on what molecules are being detectedsample, but all act via binding the sample based on its polarity. Next, the samplees the column and is detected by either a UV or fluorescence detector, which sendsdata to a computer. The purified sample can then be collected and saved for othereriments, or it can go to waste.

293Reverse-phase HPLC Analysis and Purification of Small Molecules

larger sample volumes and amounts of material. Columns vary by particle

size, length, internal diameter, and the chemistry of the packing material.

Silica is the most commonHPLC column-packing component, and it is fre-

quently derivatized with hydrophobic alkyl chains (–CH2–CH2–CH2–

CH3) that interact with the sample. There are three common chain lengths,

C4, C8, and C18, with C4 used for proteins and C18 generally used to cap-

ture peptides or small molecules. C30 columns have been used to analyze

carotenoids, plant compounds that are yellow, orange, and red, and are often

associated with the anticancer properties of foods such as tomatoes. There

are multiple types of detectors, which are needed to ‘see’ the sample as it

leaves the column; fluorescent and UV detectors are the most commonly

used. The data from the detector are sent to a computer where the software

gives you a visual interpretation of the elution profile. The sample then

leaves the HPLC system and can go to waste, a mass spectrometer (LC-

MS), or be collected for another experiment.

The benefits of HPLC include that analysis can be done quickly and with a

high degree of resolution. Columns can be reused, resulting in analyses that are

highly reproducible. If you can afford the more expensive machinery, both

instrumentation and quantification can be automated, which saves a lot of time

in the lab. A downside toHPLC is that it takes quite an investment ofmoney in

machinery toget thismethodologyupand running ina laboratory.HPLCis still

not a high-throughputmethodology, and if you have dozens or even hundreds

of samples, it will also take an investment of time to get results.

2. EQUIPMENT

Savant Automated Environmental SpeedVac® System (AES1010)

Vortex mixer

Water bath

Solvent degasser

Precolumn

Analytical or preparative C30 column (4.6 mm�150 mm, 3 mm; YMC,

Wilmington, NC)

HPLC system, which should include:

Injector: automated or manual

Pumps: Rainin Dynamics gradient pump (model SD-200; Varian,

Walnut Creek, CA) or Prostar pump (model 210; Varian)

Optical detector: photodiode array detector (model 2996; Waters,

Milford, MA)

Millennium32 software (Waters)

294 Kirstie Canene-Adams

Surgical scissors

Hamilton syringe

Glass tubes, 50 ml

Micropipettors

Micropipettor tips

1.5-ml polypropylene tubes

3. MATERIALS

Butylated hydroxytoluene (BHT)

Echinenone

Potassium hydroxide (KOH)

Argon gas

Ammonium acetate (NH4OAc)

HPLC grade ethanol

HPLC grade methanol

HPLC grade hexane

HPLC grade water

HPLC grade methyl tertiary butyl ether (MTBE)

3.1. Solutions & buffersStep 1 Saturated KOH

Dissolve 120 g KOH in 100 ml distilled water. The solution will heat up as the

KOH is added. If needed, add more KOH until it falls out of solution

Step 3 1.5% Ammonium Acetate

Component

Final concentration Amount

HPLC grade water

1 l

Ammonium acetate

1.5% 15 g

Carotenoid Mobile Phase ‘A’

Component

Final concentration Stock Amount

Methanol

83% 100% 830 ml

MTBE

15% 100% 150 ml

Ammonium acetate

0.03% 1.5% 20 ml

295Reverse-phase HPLC Analysis and Purification of Small Molecules

Carotenoid Mobile Phase ‘B’

Component

Final concentration Stock Amount

Methanol

8% 100% 80 ml

MTBE

90% 100% 900 ml

Ammonium acetate

0.03% 1.5% 20 ml

Caution

Take extreme care when making and working with saturated KOH: POISON!

DANGER! Corrosive! Causes severe burns to skin, eyes, respiratory tract, and

gastrointestinal tract. This material is extremely destructive to all body tissues.

May be fatal if swallowed, harmful if inhaled. Consult the MSDS for proper

treatment for any contact.

4. PROTOCOL

4.1. Preparation

Samples can be of human, rodent, plant, or dietary origin and should be

stored as appropriate for the molecule being analyzed via HPLC. In the

example presented here for carotenoid analysis, samples should be stored

at �80 �C for long-term storage. The extraction protocol will vary greatly

depending on yourmolecule of interest, and this protocol uses the extraction

protocol for carotenoids.

4.2. Duration

Preparation

About 3–5 h

Protocol

About 5–8 h

Time will depend greatly on the number of samples!

See Fig. 23.2 for the flowchart of the complete protocol.

5. STEP 1 BREAK DOWN THE MATRIX OF THE SAMPLESTHAT CONTAIN THE MOLECULE OF INTEREST

5.1. Overview

This first step of the carotenoid extraction procedure will break down the

matrix of the food, drug preparation, or tissue to allow the carotenoids to

Figure 23.2 Flowchart of the complete protocol, including preparation.

296 Kirstie Canene-Adams

be freed into solution. This protocol has been extensively refined and the

optimum extraction conditions were published by Lu et al. (2008) for the

extraction of tomato plant cells, but there is a range of conditions that could

work, depending on the tissue to be extracted.

5.2. Duration1 h

1.1 Add 1.56–5 ml of ethanol with 0.1% BHT to each sample in a 50-ml

glass test tube.

1.2 Mince tissue with scissors or homogenize it for 28 s using a tissue

homogenizer.

1.3 Place the samples on ice.

1.4 Add echinenone as the internal standard.

1.5 Add 0.29–1 ml saturated KOH.

1.6 Vortex well.

1.7 Place samples in a water bath at 60 �C for 30 min.

1.8 Vortex the samples every 10 min.

5.3. TipBHT is used as an antioxidant to protect the carotenoids from degradation once it is

removed from the original sample matrix. Samples are also placed on ice to avoid sam-

ple degradation.

297Reverse-phase HPLC Analysis and Purification of Small Molecules

5.4. TipMake sure to cut tissue into small pieces, about the size of half of a grain of rice so that

the KOH can degrade the whole tissue for proper extraction.

5.5. TipEchinenone is used as an internal standard to be compared with the standard curve and

to serve as a marker for any degradation of the carotenoids that may occur during the

procedure since it degrades in a similar fashion.

5.6. TipIn addition to using echinenone as an internal standard, prepare a standard curve with

a range of echinenone concentrations including both the lowest and highest levels of

your compound of interest.

5.7. TipThe amount of echinenone added to each sample depends on the source of the sample

being analyzed. For example, the liver contains much higher levels of carotenoids than

the brain, so more echinenone is needed in the liver sample. The amount of the internal

standard should be within the expected concentration range of the compound in a given

source. If the range is 100–1000 units, you should include a range of 10–1100 units

of the internal standard. However, you would not want to include 1000 units of the

standard in a sample containing only 10 units of the compound or your internal stan-

dard peak will dwarf the HPLC peak of interest. If you do not know the expected

concentration range, reference the literature or perform a couple trial runs with different

amounts of sample and internal standard.

See Fig. 23.3 for the flowchart of Step 1.

6. STEP 2 EXTRACTION OF CAROTENOIDS FROMTHE SAMPLES

6.1. Overview

These steps will remove the carotenoids from the plant, human, or rodent

matrix. The samples will be extracted using hexane so that the carotenoids

can cleanly enter the HPLC system.

6.2. Duration2 h

2.1 Add 2 ml water to each sample.

2.2 Add 2.5–6 ml hexane to each sample.

Figure 23.3 Flowchart of Step 1.

298 Kirstie Canene-Adams

2.3 Vortex for 17.5–30 s.

2.4 Transfer the top hexane layer to a clean test tube, and repeat Steps

2.1–2.3 twice, for a total of 3 times.

2.5 Dry the samples down in a SpeedVac®.

2.6 Cover the samples with argon gas, store in �20 �C freezer, and run

them on the HPLC system within 48 h.

6.3. CautionWork with hexane in a fume hood to avoid inhalation.

6.4. TipArgon gas is an inert gas that is used to remove the oxygen and any additional mois-

ture from the sample to prevent oxidation and degradation until it is run on the HPLC

machine.

See Fig. 23.4 for the flowchart of Step 2.

Figure 23.4 Flowchart of Step 2.

299Reverse-phase HPLC Analysis and Purification of Small Molecules

7. STEP 3 CAROTENOID ANALYSIS ONA REVERSE-PHASEHPLC-PDA SYSTEM

7.1. Overview

The purpose of this step is to quantify the amount of carotenoids in a sample

using an HPLC system. As published by Lu et al. (2008), this HPLC system

consists of a Rainin Dynamics gradient pump (model SD-200), a Prostar

pump (model 210), a C30 column with a precolumn, a photodiode array

detector (model 2996), and Millennium32 software.

7.2. Duration5–8 h depending on the number of samples

3.1 The Mobile Phase A and Mobile Phase B consist of various levels of

methanol, MTBE, and ammonium acetate aqueous solution. Program

the gradient procedure at a flow rate of 1 ml min�1 is as follows:

5 min hold at 10% B

12 min linear gradient to 65% B

12 min linear gradient to 95% B

5 min hold at 95% B

2 min linear gradient to 10% B

2 min hold at 10% B (for a total time of 38 min)

Figure 23.5 Flowchart of Step 3.

300 Kirstie Canene-Adams

3.2 Set the UV detector to 472 nm with the column at room temperature.

3.3 Inject a single sample at a time using a Hamilton syringe, and run the

HPLC using the gradient flow of the two mobile phases as described

above; the software controls the flow of the mobile phases.

3.4 Sample runs should be performed in duplicate and averaged.

3.5 Analyze data based on the procedure of the software manufacturer.

7.3. TipRemember to degas the mobile phases to avoid introducing air bubbles into the HPLC

system as this will result in false readings.

7.4. TipFor reproducible runs, ensure that sufficient time is allowed for reequilibration of the

column between sample injections.

See Fig. 23.5 for the flowchart of Step 3.

REFERENCESReferenced LiteratureLu, C., Engelmann, N. J., Lila, M. A., & Erdman, J. W., Jr. (2008). Optimization of lycopene

extraction from tomato cell suspension culture by response surface methodology. Journalof Agricultural and Food Chemistry, 56(17), 7710–7714.

301Reverse-phase HPLC Analysis and Purification of Small Molecules

SOURCE REFERENCEShttp://www.sigmaaldrich.com/analytical-chromatography/analytical-products.html?

TablePage¼22679949, Sigma-Aldrich HPLCMethods Guide, Accessed April 19, 2010.

Related Literaturehttp://ccc.chem.pitt.edu/wipf/Web/LCMS%20trouble%20shooting.pdf, Waters Trouble-

shooting Guide, Accessed April 19, 2010.http://www.waters.com/waters/nav.htm?cid¼10048919&locale¼en_US, Waters HPLC

Introduction.