extraction of hops acids from the hops plants

Hops acids were extracted from the plants using a specific process. The hop plant components are absorbed by the solid phase using a C18 column. Following washing, methanol was used to desorb the hop chemicals. The quantity of hop acids present was quantified using high-performance liquid chromatography. HPLC proves to be an alternate method to measurements of and acids in hops plants, despite the fact that it does not provide 100% precision. Before applying any analytical procedure to quantify the concentrations of the analytes in the sample, SPE is used to clean up the sample. The stationary phase used in the liquid chromatography column is also used in the solid phase. Solid phase extractions procedure is useful in extracting the organic compounds from the plants and environmental sample, and also removes interfering complex matrices to obtain the analytes of the interest. Additionally, SPE is helpful in the isolation of analytes from purified extract and liquid form. This paper will discuss the efficiency of the procedure of SPE over the LLE when analyzing the hops α and β acids from the hops plants.







Introduction

Preparation of the sample is vital in the analytical analysis of the sample. According to the estimation, around 80% of the typical total analysis time is taken in the transformation of the bulk sample to a suitable form of analysis (Mitra 31). The most critical point in the sample preparation is the quantitative extraction of the analytes (compounds of the interest) from another matrix (components of the sample) which interferes with an instrumental analysis in analytical chemistry (Mitra 33). Traditionally, there has been an extensive use of the liquid-liquid extraction of the analytes in the experimental samples. However, due to the development, solid phase extraction techniques are a handy tool as an alternative technique to liquid-liquid extraction. During the solid phase extraction, the liquid sample passes through a solid phase where the analytes have a higher affinity than the bulk liquid (Buszewski and Malgorzata 199). Subsequently, the sorbent selectively retains the analytes and extraction occurs through elution using appropriate solvent. Thus, there is a removal of the many sample matrixes through the solid phase extraction. Though the solid phase was applicable experimentally in the 1940s, 1970s was the start of the development of the process leading to its adoptions and widespread into the current analytical method (Mitra 40). Initially, the primary use of the solid phase extraction was a concentration of small amount of the organic pollutant from the water (Nevado et al. 503). However, currently, its applications are spread to a wide variety of matrices such as milk, oil, sediments, blood, serum, soils, plant and animal’s tissues, pharmaceuticals preparation and plasma (Sun et al. 848).

Hops plant contains essential oils typically 0.5-0.3% by mass (Nevado et al. 499). The oil is made up of a primary class of compounds referred to us as terpenes. Recently, there is discovery of the presence of disulphur compounds in hops (Neve 10). The sulfur compounds present are thiol and thioesters compounds in hop plants (Neve 12). Similarly, the plants contain polyphenols which are water-soluble compounds. Proanthocyanidins, polyphenol class, are present in large quantities and very crucial, especially in the beer flavor. The monomers of the polyphenols include catechin, epigallocatechin, gallocatechin, and epicatechin. β-glycosides glyconjugation are another vital aroma extracts from the hops plants (Neve 13). Glyconjugation allows hop plants to produce and store volatile compounds in a soluble and inactive state. Glyconjugation is also an essential mechanism that helps in transport and continuance synthesis of volatile aroma compounds in situ, especially when synthesizing against an increasing concentration gradient (Nevado et al. 504). Hops plant contains hundreds of the components as mentioned earlier. However, of particular interest are the hops α and β-acid. Hops α and β acids have slow solubility especially in beer, and therefore it had limited possibility during the dry hopping (Neve 10).

Analysis of the bitter principles in beers requires liquid-liquid extraction or SPE extraction followed by the HPLC analysis (Sun et al. 849). Unfortunately, using liquid-liquid extraction proves to consume a lot of organic solvent, lack of reproducibility and only main compounds such as iso-humulones and humulones can be monitored using the methodology (Sun et al. 851). Hop aroma extraction is quantifiable using headspace solid phase extraction and GC-FID analysis, while non-volatile extraction measures with HPLC or spectrophotometry (Buszewski and Malgorzata 203).

Alpha acid (humulone, cohumulone, and adhumulone) and β acids (lupulone, colupulone and adlupulone) are the precursors of the bitterness of the beer (Neve 18). There is acyloin-type ring contraction during the hop boiling; a thermal isomerization converts the α acid to iso-β -acids. Therefore, each α acid gives to two epimeric iso-α acids and hence, conversion of the α acids results to six major iso-α-acids (Laws et al. 187).

The principal modes of the solid phase extractions are normal, reversed or ion exchange. It is worth noting that the method differs on the affinity of retaining the analytes or the compound of interest. Normal phase solid phase extraction uses solid phase to extract polar compounds from the nonpolar sample matrix (Sun et al. 853). To elute the analytes, it is imperative to use solvent that is more polar than the sample’s original form. The importance of the reversed phase is that it removes the nonpolar analytes from a polar matrix. Usually, the organics are useful for elution while the hydrophobic solid phase is used to retain the analytes. When dealing with the charged molecules in solutions, ion-exchange is the best selection. In the ion exchange extraction, there is adjusting of the sample PH before the extraction, and an organic solvent is used for the elution.

Solid phase extractions sorbents are available in a variety of formats: cartridges or columns similar to disk, bulk or syringe barrels (Buszewski and Malgorzata 204). Typical sorbents depend on the functionalized silica or polymers, bio sorbents, carbon nanotubes and nanoparticles. Column housing consists of polypropylene or glass with the sorbents contained by the Teflon frits, stainless steel or polyethylene. When selecting appropriate solid phase extraction equipment and mode, there should be consideration of the analytes concentration, matrix, and sample volume. Extraction can be done using individual’s columns, vacuum flask assembly, large volume samplers or vacuum accommodating 12-24 samples (Wan Ibrahim et al. 234).

Typically, there are four methods for the solid phase extraction; sorbent Conditioning, sample loading, washing, and elution. Therefore, after the elution, elute is ready for the instrumental analysis. Sorbent conditioning step prepares the sorbent by making it compatible with the liquid solution, removing any impurities or contaminant and promoting proper surface contact. Usually, volume of 5-60 ml of solvent is enough for the sorbent in an SPE disk or tube (Wan Ibrahim et al. 236).

After the conditioning, there is quantitatively transfer of the sample to the column and allowing it to pass through using the pump, vacuum or applied pressure. The flow rate of the sorbent depends on the column dimensions, analytes and the particle size of the sorbent. Although the dropwise is ideal comprising the flow rate of 2-50 ml/min, in the entire cases, the flow rate should be constant (Wan Ibrahim et al. 238). The undesired matrix passes through the column while retaining component of interest (Wan Ibrahim et al. 239). After the passing of the sample through the column, there is column washing using appropriate solvent. The process is essential to remove the undesired matrix while retaining the analytes. Volumes of the 5-60 ml of solvent for the SPE disks or tube are used for the wash stage. The recovery of the analytes is the final step of the extraction process. And so, the analytes move out from the sorbents and return to the liquid phase ready for the analysis while the undesired matrix trapped remains behind. Solvent uses a small volume to extract the analytes from the solid phase fully. Usually, 200 microliters to 10 ml of the organic liquid pass and collects to the column (Wan Ibrahim et al. 240).





Method

Chemical and reagents

Methanol (HPLC grade)

N-hexane

N-pentane (HPLC grade)

Dichloromethane (HPLC grade)

Ethyl acetate (HPLC grade)

ENV and Sep Pak plus C18 cartridges

Chloroform

Sodium chloride (NaCl)

Water

Acetonitrile

Citric acid buffer

50% sodium hydroxide (NaOH)

Crystalline dicyclohexylamine (DCHA) salts of trans-iso-α-acids





Optimization of SPE and eluting solvent

There were two studies on different modes of a solid phase (Hydroxylated polystyrene-divinylbenzene copolymer (ENV+) and reversed solid phase (c18)) (Buszewski and Malgorzata 207). 2ml of each eluting solvent was used to recovers the hops acids from the solid phase sorbents. N-hexane and N-pentane compounds were the worst sorbents eluting solvents. Methanol was the best eluting solvent for compounds in Sep Pak plus C18 cartridges (Wan Ibrahim et al. 243). The highest isolation and concentration efficiency were through the use of the dichloromethane as eluting solvent.

Optimization of LLE and extracting solvent

Chloroform, dichloromethane, n-hexane and ethyl acetate were used for complete extraction. N-hexane proves to be the worst LLE solvents while chloroform, ethyl acetate, and dichloromethane provide the target compounds. The highest isolation was through the use of the dichloromethane as an extracting solvent.

Procedure

Liquid-liquid extraction procedure

200 ml of a sample containing 4grams of NaCl was placed in a 250 ml glass flask. The extraction was performed with 5ml of ethyl acetate, N-hexane, chloroform, and dichloromethane. The vial was introduced into the ultrasonic bath and sonicated for 30 minutes at 240C. The organic layer was then separated by pipetting. All samples were extracted in duplicate, and finally, the sample was injected into the HPLC instrument.



Solid-phase extraction procedure

Two different solid phase cartridges (isolate ENV+ and Sep Pak plus cartridges) were tested for the concentration and isolation of analytes from the hops plants. The process of the solid phase extractions was in SPE vacuum manifold (12-port model) (Wan Ibrahim et al. 245). The cartridges were placed in the manifold systems and activated with 4 ml dichloromethane, 4 ml methanol and rinsing 4ml of water. The 100 ml samples were passing through the cartridges by vacuum manifolds, after which the sorbents were, dried (Wan Ibrahim et al. 246). The analytes were eluted from the cartridges using an organic solvent (dichloromethane, n-pentane, methanol, and dichloromethane)

HPLC instrumentation

The entire chemicals are purchased from reliable suppliers with high purity including acetonitrile, methanol, and crystalline dicyclohexylamine (DCHA) salts of trans-iso-α-acids. DCHA contains 66.5%w hop acids. The extracts of iso-α-acids and β-acids are from hops plants (Neve 14).

HPLC conditions: the HPLC consist of an HP 1090 with a photodiode array detector. The detector was set to measure the absorbance of iso-α acids at 275nm and α and β acids at 314nm; the column temperature was set at 400C, injection volume was 20ul. For the complete separation of the mixture in the components, the mixture A was acetonitrile: methanol: citric acid buffer in the solution with a ration of 17:25:57(v/v/v). Mixture B was citric acid buffer: acetonitrile with a ratio of 45:55(v/v). There was an adjustment of the citric acid buffer to pH 7 with 50% NaOH solution and filtering before the combination with the organic solvent.



Results



Figure 1 above: (Buszewski and Malgorzata 200)

The solid phase extraction method uses C18 SPE cartridge and with an elute mixture of methanol, acetonitrile, water, citric acid, which are easy to handle and demonstrate to be a rapid technique. Liquid-liquid extraction shows problems in reproducibility and more sample matrix. Solid phase extraction shows excellent resolutions and peaks shape as shown in the figure above (Buszewski and Malgorzata 200). SPE has decreased organic solvent usage and waste generation. SPE has a higher and more reproducible recovery comparing with the LLE. SPE has no emulsion formation and has a cleaner extract (no contamination or solvent impurities). The SPE has tunable selectivity, and it is readily automated.

Each peak represents the response of a detector to different compounds. On injecting the sample, the chromatograms start to appear with a straight line (baseline), which signifies the mobile phase. The SPE shows satisfactory resolution since there is disengagement of the adjacent peaks in two maxima (Neve 53). LLE shows poor chromatograms because of early peaks which are broad. SPE shows excellent chromatograms because of the late-appearing peaks which are very narrow. Resolution is an ability to separate two peaks (Neve 55). In the SPE, there is higher resolution of the peak while in LLE displays a reduced resolution of the peaks. The migration rate of the analytes in chromatogram method is the relative affinity of the substance to mobile phase or stationary phase. SPE indicates good peaks because of its symmetrical nature or Gaussian peak. LLE shows poor peaks shapes because some of the peaks are tailing and fronting. SPE illustrates less tailing, high efficiency, narrow peak width, improved resolution, and more accurate quantitation. LLE shows less efficiency, broad peak width, and low resolution (Neve 59).

Discussion

It is worth noting that before the extraction, samples pretreatment such as filtration, additional of organic solvent or adjustment of the pH is vital since it enhances the retention of the analytes in the solid phase. However, the types of the sorbent, sample volume, matrix, and analytes determine the sample preparation (Mitra 43).

Solvent extraction methods use non-polar solvents which are miscible with water to extract the analytes using the significant solubility of the compound of interest in the solvent than water. Volatile solvents such as benzene, ether, ethyl acetate, hexane, and dichloromethane are useful for the extraction of the semi-volatile compounds from the water. Ethyl acetate and ether are suitable for the extraction of the relatively polar compounds containing oxygen. Hexane is appropriate for the extraction of the non-polar compounds such as aliphatic hydrocarbons. Benzene is ideal for extraction of aromatic compounds such as phenols compounds in hops plants (Neve 45). Dichloromethane has higher extraction efficiency for a wide range of polar to non-polar compounds. Simultaneous analysis of the dichloromethane is due to its low boiling points and easy re-concentration after the extraction. In addition, dichloromethane has higher specific gravity, and therefore it is easy to separate from water. Lastly, dichloromethane is non-flammable. Recently, there is refraining from the use of benzene due to its carcinogenic nature in the liquid-liquid extraction.

Changing pH of the sample can sometimes change the character of the sample. For instance pH of the less than 2, there is sufficiently ionization of the basic compounds, and there is no extraction allowing extraction of neutral and acidic compounds (Wan Ibrahim et al. 251). Sometimes salting out techniques is applicable when extracting compounds which are relatively soluble in water due to increase in extraction rates. Addition of the salts assists in decreasing the solubility of analytes and solvation power of the sorbents. Therefore, salting is useful in both the liquid-liquid extraction and solid phase extraction techniques (Wan Ibrahim et al. 252).

Extraction is achievable by shaking the solvent and the water sample in separating funnel. However, it sometimes difficult to separate the solvent from the aqueous phase due to the formation of a large number of emulsions. To overcome the challenge, an addition of the methanol and sonication of the mixture in an ultrasonic bath helps to disperse the emulsions. Additionally, continuous liquid-liquid extraction methods help to circulate solvents in unique glassware. Although the continuous way has extraction efficient, it is not appropriate for the thermally unstable compounds due to long duration extraction.

Solid phase extraction has several advantages over liquid-liquid extraction that has led to its rapid development and increasing usage as sample preparation techniques (Mitra 44). The benefits include the reduced solvent use, higher concentration factors, and faster and fewer labors intensive sample manipulation (Nevado et al. 497). There is accommodation of large volume of the samples and multiple extractions can co-occur as well as an automation of the process. The easiness and rapid of the process makes it desirable and alternative to the liquid-liquid extraction. On the other hand, liquid extraction automation is insignificant and challenges when it comes to labor manipulation of the sample.

Secondly, the solid phase uses a reduced or minimal solvent (Wan Ibrahim et al. 252). Apart from the cost arising from the purchase of bulk of the solvent, liquid-liquid extraction pose issues regarding its disposal mechanism. Liquid-liquid extraction has a potential for contamination of the sample extracts and analyst exposure to hazardous chemicals.

Thirdly, usage of the small elution volumes during extraction from the solvent reveals that concentration of the analytes is achievable. Liquid-liquid extraction concentration factors are limited to the volume ratio of the solvent and sample. Liquid-liquid extraction can achieve high efficient concentration factors of up to 100 while solid-phase extraction can attain up to 1000 (Buszewski and Malgorzata 208). Even though solid phase is the best method for the sample preparation, in recent years, it is useful as pre-concentration procedures in other analytical procedures (Mitra 46). For instance, the capillary electrophoresis is useful analytical method since it provides a high-resolution separation with small volumes (Maijó et al. 2114). However, with relatively dilute samples, it gives a low sensitivity. When coupling the capillary electrophoresis with solid extraction, it allows for the simultaneous concentration (Maijó et al. 2116) Also, clean-up of the large sample volumes before the injection on the capillary electrophoresis instrument results in lower detection limits and improved sensitivity (Nevado et al. 502). Therefore, SPE application is mainly in the capillary electrophoretic analysis of acidic pharmaceuticals in the river, determination of insulin derivatives in biological fluids, determination of the melamine in milk, detecting of the amine, and more so in antioxidants in olive oil (Sun et al. 852).

Conclusion

Solid phase extraction is a perfect alternative to the liquid preparation for determination of α and β acids in hop products. In comparison with the liquid-liquid technique, solid phase has improved precision and reduction in cost and time (Buszewski and Malgorzata 210). Due to the complexity of hops acids and low concentration of acids, its analysis requires isolations or pre-concentration steps (Neve 51). The low concentration of compounds makes enrichment necessary as the basis for identification and quantification of liquid-liquid and solid-phase extraction (Nevado et al. 497). The method aims at selecting the finest extraction techniques for studying the composition of hops plants using LLE and SPE, in quantitative determination of nonvolatile and volatile compounds in hops plants. Solid phase extraction appears an alternative to liquid-liquid extraction because of low cost, easy automation, simplicity, highly purified extracts and high recovery of the analytes. Additionally, in SPE there is analytes concentration, compatibility with instrumental analysis, simultaneously extract analytes of wide polarity range and reduction in an organic solvent (Wan Ibrahim et al. 253). Currently, the laboratory cannot do without the technique and therefore it is a robust method for the sample preparation. The processes are critical in sample preparation techniques for the concentration, isolation, medium exchange and clean-up (Mitra 48).

Works cited

Buszewski, Boguslaw, and Malgorzata Szultka. "Past, present, and future of solid phase extraction: a review." Critical Reviews in Analytical Chemistry 42.3 (2012): 198-213.

Laws, DEREK RJ, NIGEL A. Bath, and JOHN A. Pickett. "Production of solventfree isomerized extracts." J. Am. Soc. Brew. Chem 35.4 (1977): 187-191.

Maijó, Irene, et al. "An in‐line SPE strategy to enhance sensitivity in CE for the determination of pharmaceutical compounds in river water samples." Electrophoresis 32.16 (2011): 2114-2122.

Mitra, Somenath, ed. Sample preparation techniques in analytical chemistry. Vol. 237. John Wiley & Sons (2004): 31-49.

Nevado, Juan José Berzas, Virginia Rodríguez Robledo, and Carolina Sánchez-Carnerero Callado. "Monitoring the enrichment of virgin olive oil with natural antioxidants by using a new capillary electrophoresis method." Food chemistry133.2 (2012): 497-504.

Neve, Ray A. Hops. Springer Science & Business Media (2012): 10-60.

Sun, Hanwen, et al. "Determination of melamine residue in liquid milk by capillary electrophoresis with solid-phase extraction." Journal of chromatographic science 48.10 (2010): 848-853.

Wan Ibrahim, Wan Aini, et al. "Application of solid-phase extraction for trace elements in environmental and biological samples: a review." Critical reviews in analytical chemistry44.3 (2014): 233-254.