Presented in partnership Presented on on i C PL ng Sample Sample ng i n H n i

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P Improv Sponsored by Sponsored J U LY 2 0 1 7 ® Sample Prep Overview

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hile sample preparation is a routine task for chromatography labs, it cannot be taken for granted. Sample prep may well be one of the most important ways of ensuring the selectivity, sensitivity, and Wreproducibility of an analytical method. In Improving Sample Preparation in HPLC (sponsored by GE Healthcare, and presented in partnership with LCGC), experts explain how labs can ensure their sample prep techniques are supporting the success of even the most advanced chromatographic methods.

First, Nicolas Snow, PhD, a professor of chemistry and biochemistry at Seton Hall University, provides an overview of how some basic factors—like lab glassware choice and solvent mixing—can all have a major effect on the reproducibility of a separation method. Snow also reviews the fundamentals of some of the most common sample preparation techniques, such as liquid–liquid extraction, solid-phase extraction, solid-phase microextraction, and QuEChERS, and the importance of keeping equilibrium and kinetics in mind.

Next, “Sample Prep Perspectives” columnist Douglas E. Raynie interprets the results of a recent LCGC North America readership survey about the use of sample preparation techniques. He examines both short-term trends (especially in the use of automation and newer instrumental extraction techniques) and long-term trends spanning over a decade.

In a separate piece, Raynie and Ronald E. Majors explore high-throughput sample preparation as a possible technique for accelerating sample preparation time. They suggest that the best approach may be a combination of methods for achieving high-throughput sample preparation.

Last, Dwight Stoll, editor of LCGC’s LC Troubleshooting column, discusses some best practices in instrument set-up related to filters and filtering, two important parts of any liquid chromatography system.

As this LCGC eBook demonstrates, a well-thought out sample preparation technique is a lab’s first step in an ensuring an accurate and reliable chromatography method—and proper planning for this stage is critical. TOC Improving Sample Table of contents Preparation in HPLC

Sample Prep Overview Sample Preparation: The Forgotten Dimension in HPLC Nicholas H. Snow 5

Sample Prep Trends Trends in Sample Preparation Douglas E. Raynie 13

High-Throughput Sample Prep Exploring the Possibilities of High-Throughput Sample Preparation Douglas E. Raynie and Ronald E. Majors 30

Filtration Filters and Filtration in Liquid Chromatography: What To Do Dwight Stoll 37 5 | July 20175 |July | LCGC Nicholas H. Snow H. Nicholas Th Sample D i men e Forgotten si on P reparat i n H basics ofbasics sample prep, it is vital that: and reproducibility.accuracy Reviewing the amethod’s that affects acritical factor be can While sample preparation is aroutine it task, SampleClassic Prep Techniques and reproducibility of an analysis. preparation can affect the selectivity, sensitivity, In this article, learn about some ways that sample important aspects of running analytical methods. of as basic chemistry, but they may be the most Sample preparation techniques are often thought Overview chromatographic methods. reproducibility from even the most advanced the most is factor important achieving in forgotten, often Although sample preparation 4. 3. 2. 1.

and kinetics. optimized using the ideas of equilibrium Sample preparation techniques are handled safely. Samples, reagents, and chemicals are according to the method’s needs. Solvents and glassware are chosen operatingstandard procedures. samples collect accordingAnalysts to PL

i C on:

shutterstock.com/Looker_Studio Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

Red = 2015 Blue = 2013

Figure 1: Popularity of sample preparation techniques. Source: D.E. Raynie, LCGC North America, 34(3), 174–188 (2016).

5. Samples are kept as clean as a form that is ready to be injected into an possible. instrument can require numerous steps, Sample preparation requires precise each with its own potential for introducing handling and manipulation of samples variance to the method. Gravitmetric steps from the time they enter the lab until the often have the fewest errors associated vial is placed on the tray. One should with them, assuming the balances are always think about which steps of the properly maintained and calibrated, and sample handling process could enable proper operating procedures are used. hidden errors to creep into the method. Dilutions, on the other hand, are often a Several techniques are used in neglected source of variance. sample preparation, ranging from well- Glassware choice. It is common established procedures like weighing, to do simple dilutions in volumetric dilution, and filtration, to relatively glassware. It is imperative to use good new technologies like QuEChERS. The techniques and avoid bad practices results from two recent surveys about that can negatively affect quantitative the popularity of sample preparation analysis. For instance, one may be techniques are shown in Figure 1. Older introducing precision and accuracy methods are by far the most common. errors into the method by using smaller The process of turning a raw sample into glassware, which is chosen for smaller

6 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

samples. Larger flasks for larger samples variance that blindly picking a stopper offer better precision, at least on a from the drawer could result in a poor fit. percentage basis. In addition, when If a leak occurs during the inversion and using nonaqueous solvents, analysts mixing step, some solvent will be lost and may also see some small errors in the concentration will be thrown off. working with volumetric glassware One way to check to see if a leak is because they were calibrated for water. present is to insert the lid and see if any Class A glassware has a known ground glass is visible. If there is, switch to tolerance that should be considered, another lid until no ground glass can be and is higher for smaller volumes. For seen. example, a 250-mL volumetric flask has a Last, glassware has a finite lifespan. The tolerance of 0.048%, while a 10-mL flask volume of a heavily used flask will drift is 0.20%. Performing serial dilutions is a over time. If the etched labeling on the good way to save on the use of solvent, side of the glassware is no longer legible, but introduces extra steps into the it might be time to consider replacing it. process. Each step comes with additional In a nutshell, one must remember uncertainty, which can accumulate. (especially when faced with The same rule applies to pipettes, with reproducibility challenges) that volumetric the smallest variance resulting from the flasks and pipettes can be the source larger volumes. The type of pipette also of small experimental errors. Such matters. Volumetric transfer pipettes errors have the potential to add up to a have the tightest tolerances. Graduated potential larger error in the method that seriological pipettes can be an order may or may not be tolerable. of magnitude worse, and syringes and Solvent mixing. Mixing aqueous and automatic pipettors are the worst. It is also nonaqueous solvents results in a change good to keep in mind that plastic pipette of volume. Whenever possible, the mixing tips have an added complication: There of two solvents inside a volumetric flask is a high surface area-to-volume ratio should be avoided or minimized. Although and a plastic surface has the possibility of it adds a step, it is better to prepare the adsorbing hydrophobic analytes. solvent mixture beforehand and then use it Glassware condition. The condition in the volumetric glassware. and arrangement of glassware are also Safety. Safety is also an important important. When flasks are shipped from consideration in any laboratory the factory, they are paired with well-fitting procedure. Proper solvent handling, such stoppers. These items invariably become as aliquoting and secondary containment, separated during cleaning and normal use. are essential. Having standard operating While it is not critical to keep a flask paired procedures (SOPs) in place for solvent with its original stopper, there is enough handling has many benefits. Not only

7 | July 2017 | LCGC Theory – Multiple

Sample Prep Sample Prep High-Throughput Filtration ExtractionsOverview Trends Sample Prep

K More extraction steps = more complete extraction

Use multiple steps if K is low

Figure 2: Multiple extractions can be performed until sufficient analyte has been extracted.

do SOPs improve safety, but they also the solution and a separate phase. In some increase the reproducibility of the cases, the goal is to capture the analyte,18 and analysis. This can include simple things in others, it is to remove interferences. For such as aliquoting the needed solvent any partitioning separation, two important from the bottle rather than pipetting parameters that must be kept in mind are directly, which can lead to contamination. equilibrium and kinetics. Solvent choices are not absolute. There LLE. LLE is simple, easily understood, are times when a slightly suboptimal and relatively inexpensive, although it is solvent is acceptable if it greatly reduces not always easily adapted to automation. the risk associated with a procedure. The figure of merit for an LLE is the Safety considerations become particularly distribution constant (K), which is defined important when working with pure as the ratio of the concentration of the standards of highly toxic compounds. analyte in the two phases at equilibrium. When extracting from an aqueous phase Phase Equilibrium and Kinetics to an organic phase, a high value of K Many sample preparation techniques are means that the analyte will preferentially separations in their own right, including partition into the organic phase while a liquid–liquid extractions (LLE), solid- low value means that the concentration phase extraction (SPE), solid-phase in the aqueous phase will be higher. If microextraction (SPME), head space the value of the distribution constant is analysis, and QuEChERS. They depend on not known, it may be useful to do a quick the partitioning of the compounds between experiment to get a rough approximation.

8 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

For most applications, the analyst is the extraction does not have to be attempting to extract the analytes from complete if it is reproducible. the aqueous sample into an organic SPE. SPE is one example where nearly phase. For this, the value of K will ideally all of the analyte can be captured in the be high, but it will never be infinite. bed. Figure 3 shows an example of an There will always be some fraction of SPE sample treatment. The sample is analyte remaining, but near-quantitative forced through the bed with either positive extractions are possible. A low value of K is pressure from above or vacuum below. not necessarily a point of failure, however, During the loading step, the solvent must as multiple extractions can be performed be weak relative to the stationary phase until sufficient analyte has been extracted, within the column. The more strongly the as seen in Figure 2. The fraction removed analytes are bound, the tighter their band is shown as a function of the number of will be at the top of the column. During repeated extractions for several values of elution, a stronger solvent is flushed K. Sufficiently complete collection of the through, thus removing the solute from the analyte is possible even with relatively column. Just as in HPLC, step gradients weak extraction conditions. The trade- can be used to elute more than one band off is analyst time, solvent use, and the into separate fractions. concentration of the resulting extract. Kinetics are an important factor in SPE. The kinetics of the partitioning must The packed beds use larger particles be kept in mind. The mixture should be than HPLC, meaning the rate of mass shaken long enough for the partitioning transfer is much slower. Driving the to occur, and given enough time to settle solution through the bed too quickly will so that the phases can reach equilibrium. result in some analyte passing completely Failure to do so can introduce substantial through before it can be captured errors. Mechanical shakers can be by the stationary phase. Sometimes, valuable as a way of ensuring the mixtures the difference between high and low are subjected to the same conditions reproducibility comes down to the each time. strength of the vacuum system used to It is important to remember that the pull samples through the SPE cartridges. distribution constant represents the When preparing samples for HPLC, the concentration, not fraction removed. obvious choice might be to pick an SPE When the ratio of the volumes of the cartridge with a similar chemistry to the two phases is high (e.g., small volume of LC column. While this kind of cleanup has organic phase exposed to a high volume value, using a chemistry that is orthogonal of aqueous phase), then a high value of K to your subsequent separation is more is needed or only a small fraction of the likely to selectively remove compounds compound will be removed. Fortunately, other than the intended analyte.

9 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep Normal Phase SPE

www.waters.com Figure 3: Example of a solid-phase extraction sample treatment. 27

One often overlooked advantage of attention to kinetics and ensure that the normal-phase or ion-exchange SPE is that fiber is in contact with the solution long the sample is eluted in a highly aqueous enough. If one is unsure, this is a good phase. When injected onto a reversed- place to consider internal standards. phase HPLC column, the sample will stack In the past 10 years, there have been on the head of the column, thus providing numerous advances in commercially a significant boost in sensitivity. Vendors available SPME systems. They work well have excellent resources available for the with small samples and are well suited for selection and use of SPE cartridges. automation and high throughput, including SPME. In SPME, the extraction phase working with 96-well plates. Because the is typically a thin layer on a fiber. The process involves partitioning the analytes phase ratio is so high that only a small out of the sample solution, dirty or particle- fraction of the solute is captured by laden samples are much less of a problem. the extraction phase. In this regimen, Pipette-tip SPE is a growing technique, in achieving reproducibility depends entirely which the sample is pulled into a pipette tip on reaching equilibrium every time and packed with the solid phase. It lends itself avoiding variations in sample matrix or well to small samples and is relatively easily laboratory conditions that could affect adapted to automation. Unlike traditional the partition coefficient. One must pay SPE, the solution must pass through the

10 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

bed in two directions, first being drawn in drugs in blood, preservatives in pet food, and then flushed out. This places somewhat and acrylamide in fried food. tighter restrictions on the need to have a high partition coefficient. An analyte that is Conclusion only weakly bound will tend to be flushed Sample preparation is very often the out again. However, this feature is arguably biggest source of variance for a method, an advantage when there are large making it the most important factor to amounts of a weakly bound interferant. In control and design well. It is often neglected addition, clogging issues are somewhat because it constitutes the cheaper, more alleviated as the head of the column is mundane part of any method. One cannot effectively backwashed each time. always rely on the column to make up for an QuEChERS. In recent years, a new insufficiently selective sample preparation. technique has become quite popular, and Likewise, even a mass spectrometer with the is known as QuEChERS, an acronym for highest resolving power is still susceptible quick, easy, cheap, effective, rugged, and to ion suppression from an insufficiently safe. In a typical application, the sample purified sample matrix. Separations of any is homogenized and extracted using kind work best with clean samples, and an organic solvent such as acetonitrile instruments require less maintenance. Well- or ethyl acetate. The organic phase is established and standardized techniques then isolated and can be dried with can minimize variance, whether day-to-day, magnesium sulfate. As a final step, person-to-person, or lab-to-lab. derivatized particles are mixed with the Many sample prep techniques are sample to selectively capture unwanted separations in their own right, whether sample components and then separated LLE, SPE, SPME, QuEChERS, or another via centrifuge. Several surface chemistries solid-phase method. Treating them as of the particles are available, including a potentially orthogonal dimension to primary secondary amines for sugars, a primary separation is a useful way to organic acids, and lipids; graphitized simplify an otherwise complex sample. carbon black for aromatic and conjugated As with all separations, being mindful of compounds such as carotenoids and both the equilibrium and the kinetics of chlorophyll; and C18 for highly nonpolar that separation is important to both the compounds. sensitivity and reproducibility of a method. Unlike many of the other methods, the Nicholas H. Snow, PhD SPE is being used as a one-way means is a professor of chemistry and biochemistry at Seton Hall University. of removing what is not wanted from the sample. The QuEChERS method has quickly been adapted to such wide- ranging applications as pharmaceutical

11 | July 2017 | LCGC Filtration should just work

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i n Sample Sample n i on trends over the past quarter century. quarter past the over trends longer at look to us allows survey current the of timing the but field, the of state current the assess we do only not Here, generation. past the over developed techniques newer adopting are others preparation, sample of methods older, to traditional on hanging are laboratories many great a (1–5). While column Perspectives” the in reported surveys periodic of subject the been have them from emanating trends the and advances These generation. past the in made been have advances significant chromatography, modern in advances behind languished has preparation sample of field the While preparation technologies on the horizon. Respondents were asked about also sample selection criteria, and problems encountered. (cartridges, plates, disks, tips), SPE chemistries, of solid-phase (SPE) extraction devices loads, sample automation, sizes, the use technologies currentlysample beingused, investigated suchsurvey trends areas in as from 1991surveys to March 2013. The the resultstechniques of with previous from arecent on sample survey preparation compares article the results obtainedThis LCGC “Sample Prep Prep “Sample

getty Images/xxxxxxxx Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

Other Energy, petroleum Plastics, polymers, rubber Inorganic chemicals Instrument design, development Forensics, narcotics Organic chemicals Biotechnology, bioengineering Agriculture, food Food, beverage Medical, biological Environmental Pharmaceuticals 0 5 10 15 20 25 30 Total respondents (%)

Figure 1: Fields of work of survey respondents in 2013 (blue) and 2015 (red) as a percentage of total respondents. The “Other” category includes flavors and fragrances, natural products, electronics, and paper chemicals.

As in previous iterations of the industry, roughly one in seven came survey, LCGC North America sent an from academia (14.7%), a similar amount Internet-based survey to a statistically came from government (16.0%), 9.3% of representative group of readers during those responding were from independent the last quarter of 2015, nearly two years analytical companies, and the balance after the most recent survey (5). The survey came from research institutes, hospitals used a similar set of questions as those and medical centers, utility companies, administered previously. Although the and others. Figure 1 compares the response rate was down from the 2013 breakdown of the fields of work for the survey (5), it is still sufficient to yield useful 2013 and 2015 survey respondents. Slight conclusions. This installment summarizes increases in the numbers of respondents the survey results and observes trends in working in the environmental, sample preparation for chromatography. biotechnology, and food and beverage areas were observed at the expense of Survey Audience and Sample Types those working with organic chemicals, The makeup of survey respondents in agriculture and food, and instrument the current survey is similar to that of design and development. the 2013 survey (5). Nearly half (48%) Meanwhile, Figure 2 presents a of respondents came from private Pareto chart of the typical sample

14 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

Other Waxes Essential oils Sediment Gases Cosmetics Oils Petroleum products Inorganic materials Air Metals Grains Soils Solvents Animal, biological tissue Fruits, vegetables Prepared, processed foods Plants Drinking water Wastewater Physiological uids Polymers, monomers Organic chemicals Pharmaceuticals 0 5 10 15 20 25 30 35 40 Total respondents (%)

Figure 2: Sample matrices analyzed as a percentage of survey respondents in 2013 (blue) and 2015 (red). The “Other” category includes beverages, pesticides, natural products, surface water, feeds, proteins, narcotics, and eggs.

types encountered by these audiences. The number of samples represented as Following the trend in the work areas of physiological (including blood, urine, survey respondents, organic chemicals; and cerebrospinal fluid), animal, and solvents; physiological, animal, and biological tissues remained high, as did biological tissues; waste and drinking fruits, vegetables, and plants. This result water; oils; and essential oils each can be explained by the emphasis of decreased in representation among modern research in “omics” and food sample types. However, no other safety. Because the choice of sample categories stood out with significant preparation approaches is at least increases. Pharmaceuticals and organic partially dependent on the physical state chemicals are the largest categories of of the sample, this was also surveyed. sample types, consistent with the 2002 Table I indicates that nearly all analysts (4) and 2013 (5) surveys. However, in encounter liquid and solid samples and relative terms, polymers and monomers there is essentially no difference between comprised the sixth most common 2013 and 2015. However, 2015 shows an sample type in 2013 and were the third increased number of samples that are most common in the current survey. gases or gels and semisolids. This is a

15 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

Table I: Sample states encountered by survey respondents

Supercritical uid extraction Sample State 2013 2015 Large–volume trace enrichment Gases 48.3% 65.7% Stir-bar sorbent extraction Accelerated solvent extraction Liquids 94.5% 94.3% Ultrasound-assisted extraction Gels/semisolids 52.5% 65.7% Solvent exchange QuEChERS Solids 87.8% 87.1% Purge and trap Microwave–assisted extraction Matrix solid–phase dispersion about 42.5% in 1996 compared with Soxhlet extraction Dialysis nearly 70% in 2015. Cell disruption Lyophilization Solid-phase microextraction Ultraltration Sample Preparation Techniques Cooling Reconstitution A combination of sample preparation Homogenization Blending techniques are typically used in treating Precipitation a sample for analysis. We surveyed the Derivatization Headspace common techniques and report their Grinding Digestion prevalence (and comparison to the 2013 Solid–phase extraction Reagent extraction survey [5]) in Figure 3. The techniques Reagent addition Concentration are listed in order of prevalence and, as Drying Mixing expected, weighing, dilution, filtration, Internal standard addition Heating centrifugation, pH adjustment, and Column chromatography evaporation top the list. Compared Sonication Evaporation with 2013, liquid–liquid extraction (LLE) Vortexing th Liquid–liquid extraction jumped from the 12 most common pH adjustment Centrifugation technique to the sixth most common, Filtration Dilution while internal standard addition, which Weighing 010 20 30 40 50 60 70 80 was seventh most common in 2013, is Total respondents (%) 12th, and concentration, which was ninth Figure 3: Sample preparation procedures currently in use in prevalence in 2013, is 15th in the current (red) and those reported in 2013 (blue) as a percentage of survey respondents. survey. Also notable was the increased use of dilution, filtration, heating, dramatic increase compared to the results ultrafiltration, cooling, pH adjustment, presented in 1996 (3). While the question reagent addition, and centrifugation, was asked slightly differently 20 years while evaporation, solid-phase ago, if we normalize the results to the extraction, derivatization, precipitation, most prevalent physical state each year, homogenization, purge and trap, and only about 17.5% of analysts encountered solvent exchange saw decreased use. gas samples in 1996, while about 70% did The QuEChERS (quick, easy, cheap, in 2015. The increase is only slightly less effective, rugged, and safe) method, stir- dramatic for gels and semisolid samples, bar sorptive extraction, and ultrasound-

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Table II: The 10 most-prevalent sample preparation prevalent techniques, listed in order of techniques reported in 2015 and their rankings in previous surveys ranking, and their rankings in the 1991, 1991 1996 2002 2013 2015 1996, 2002, and 2013 surveys (1,3–5). Weighing 1 2 1 1 1 Dilution was apparently not surveyed in Dilution — 3 4 4 2 1991, but has consistently been among the Filtration 2 1 3 2 3 top four techniques in subsequent surveys. Centrifugation 7 8 9 3 4 Vortexing was also not surveyed in 1991 pH adjustment 9 4 2 6 5 Liquid–liquid 6 11 12 12 6 and slowly climbed to its current seventh extraction position. Sonication, which was also not Vortexing — 17 14 8 7 surveyed in 1991, has been in the 10 most Evaporation 4 5 7 5 8 prevalent techniques since 2002. On the Sonication — 36 8 10 9 Column 12 9 6 13 10 other hand, internal standard addition (third chromatography in 1991) and concentration (fifth in 1991) were not in the top 10 in the current survey Table III: Number of sample preparation techniques th th required per sample, reported by percentage of survey (12 and 15 , respectively). Drying and respondents 2002–2015 grinding were among the top 10 techniques Number of Techniques 2002 2013 2015 used in 1991 (drying 8th and grinding 10th), 1 19.3 16.8 17.4 but are 14th (drying) and 19th (grinding) in 2 28.6 31.6 18.1 3 20.7 20.5 26.8 2015. Solid-phase extraction (SPE) was 4 15 10.1 13.8 not included in the 1991 survey question, 5 8.6 9.4 11.6 but consistently was the 10th or 11th most 6 2.1 4.4 3.6 common technique in subsequent surveys 7 0.7 1.7 2.9 and fell to 17th in this survey. Although there More than 7 5 5.4 5.8 were undoubtedly differences between assisted extraction joined the survey these surveys, the general long-term choices and these techniques are seeing trends since 1991 are still valid and likely use by 5–10% of survey respondents. reflect the increased analytical emphasis It is interesting to note that each of in biotechnology, health care (including the techniques for which movement in pharmaceuticals), bioenergy, and food usage was observed involve the use of science over the past 25 years. liquid samples or liquid extracts of solid These surveys give a snapshot of samples. Meanwhile, the newcomers to the current use of sample preparation the survey (especially QuEChERS and techniques, and it is assumed that the ultrasound-assisted extraction) can be survey respondents are representative of directly applied to solid samples. the global users of these technologies. That While short-term trends are interesting, being the case, if one turns to the literature it is equally important to look at longer for guidance, does the literature provide term trends. Table II lists the 10 most an adequate reflection of actual use as

17 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

indicated by the LCGC survey? To address and SPME. Other techniques also receiving this concern, I performed a quick keyword mild attention were accelerated solvent search on selected extraction techniques extraction, evaporation, homogenization, using Sci-Finder. The results presented internal standard addition, large-volume in Figure 4 are normalized to the use of trace enrichment, matrix solid-phase SPE. While a strong correlation (correlation dispersion, microwave-assisted extraction, coefficient = 0.6798) is found between solvent exchange, SFE, and ultrasound- the literature and survey results, these assisted extraction. These plans seem to trends are not immediately apparent both address changes in the analytical upon first looking at the data. With the challenges because of emerging sample exception of solid-phase microextraction types and indicate a maturation of once- (SPME), for each technique the relative emerging technologies. literature reports lag the use by In nearly every analysis (or 82.6% of survey respondents, sometimes quite analyses according to the 2015 survey), dramatically. This lag may be due to the more than one pretreatment step is reality that, by definition, articles in peer- needed. This ignores, for example, reviewed journals are research-based, direct injection of gaseous samples or rather than reflecting more routine use of direct injection methods used with mass these methods. Additionally, the trends spectrometry (MS). Table III reports the observed in Figure 4 may be indicative number of sample preparation techniques of the service, or enabling, role played required per sample in the three most by extraction in support of analytical recent surveys. In 2002 and 2013, 52% research. of respondents were reported to use Looking at future use, Majors reported three or more techniques per sample (5) that SPME, microwave-assisted (4,5). That number has jumped to 65% extraction, accelerated solvent extraction in the current survey. My estimation is (also called pressurized fluid [liquid] that approximately 3.5 techniques are extraction), SPE, headspace sampling, used per sample, up from about three and supercritical fluid extraction (SFE) per sample previously. This is logical were expected to see increased use, as most samples will be measured by according to the 2013 survey respondents. mass or volume, extracted, and then Looking at the current survey results, have their solvent volume adjusted. only microwave-assisted extraction Additional manipulations like cleanup, and headspace sampling actually saw pH adjustment, addition of internal increased use, and SPE remained steady; standards, and so forth, add to the the other techniques saw slightly less use. sample treatment. Recalling that the great In the 2015 survey, respondents expressed majority of survey respondents handle plans to use derivatization, QuEChERS, both solid and liquid samples and two-

18 | July 2017 | LCGC Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

Supercritical uid extraction

Stir-bar sorbent extraction Accelerated solvent extraction

Ultrasound-assisted extraction

QuEChERS

Purge and trap

Microwave–assisted extraction

Matrix solid–phase dispersion

Soxhlet extraction

Solid-phase microextraction

Solid-phase extraction 0 20 40 60 80 100 120 140 160 Total respondents (%)

Figure 4: Use of selected extraction techniques as reported in the literature (red) and by survey respondents (blue), normalized to the use of SPE.

thirds also explore gases and semisolids require particle size reduction or drying (see Table I), we also looked at the before extraction, in addition to similar number of sample preparation techniques sample pretreatments used with liquid per sample depending on the physical samples. state of the sample. An estimated four techniques per sample were used for Sample Loads solids, slightly more than three techniques In these times of increased cost scrutiny per liquid sample, and almost one and in industry, it is no surprise that nearly a half techniques per gaseous sample all survey respondents predict their were used. For gaseous samples, every workload will increase or stay the analyst used four or fewer techniques same. In the current survey, 54% of per sample, 96% used three or less, and the respondents indicated that their a majority (54.2%) used only one. For sample loads will increase in the next liquid samples, about two-thirds (64.5%) two years. I believe this is the first time of survey respondents used between two in the history of these surveys that this and four techniques per sample and with has topped 50%. In the 2013 survey (5), solids, nearly the same number (63.5% 48% made this claim. Meanwhile, 45% of of survey respondents) used three to respondents in the present survey believe five techniques per sample. With gases, their workload will stay the same and direct injection following sampling is the 1% expect a decreased workload. How norm. Solids, on the other hand, generally does this look in terms of the number of

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Table IV: Number of sample preparation techniques Sample Characteristics required per sample, reported by percentage of survey Initial Volume of Liquid respondents, broken down by sample state Number of Techniques Solids Liquids Gases and Gaseous Samples 1 7.7 11.3 54.2 We surveyed the initial sample volume 2 9.6 24.2 20.8 separately for liquid and gaseous 3 34.6 22.6 20.8 samples. As we observed, 94% of survey 4 13.5 17.7 4.2 respondents dealt with liquid samples. 5 15.4 12.9 0 The trend toward smaller sample volumes 6 5.8 3.2 0 continued. Majors summarized that in 7 5.8 1.6 0 More than 7 7.7 6.5 0 1996 (5) only 5.5% of the respondents had <1 mL of total sample and that number increased to 18% in 2002 and 27% in Table V: Number of samples analyzed per instrument per week, reported by percentage of survey respondents in 2013. According to the 2015 survey, that 2002–2015 number rose slightly to 30%. This trend Number of Samples 2002 2013 2015 toward smaller samples is likely because <20 33.2 30.7 20.3 of the limited sample size of biological 21–50 27.5 30.7 27.5 fluids as well as improvements in method 51–100 21.8 19.8 20.3 101–150 7.0 8.0 11.6 detection limits and sensitivities, which 151–200 2.8 2.4 5.8 now accommodate smaller samples. >200 7.7 8.5 14.5 Looking at larger samples, the 2013 survey (5) noted 8% of respondents had samples per instrument? This question sample volumes >100 mL, down from the was asked beginning in 2002 (4) and the historical average of around 21%. In the results since then show a trend toward present survey, this number rebounded to more samples per instrument per week. 16.5% of survey respondents. While nearly half (48%) of respondents Shifting to gaseous samples, reported to the current survey report 50 or fewer for the first time in this survey, 38% samples per instrument per week, of respondents had initial sample 14.5% run more than 200 samples per volumes of <1 mL (29% were less instrument per week. Using the midpoint than 0.5 mL) and another 38% of of these ranges, a very crude estimate respondents reported sample volumes for the average is slightly more than of 1–20 mL. These small volumes are 80 analyses per instrument per week. likely concentrated aroma compounds Increased workloads will be reflected in including essential oils, flavors, and increased use of automation in sample fragrances. On the other hand, 14% preparation and chromatography, as well of respondents claimed initial sample as movement toward faster extractions volumes of 500 mL to 1 L of gas. This and chromatographic separations. seems more in line with air pollution

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studies and similar investigations with to carefully and quantitatively concentrate environmental samples. the sample volume down to 100 µL (quite the challenge, I suspect, for many of us) Weight of Solid Samples and we use standard chromatographic Previously, there was no clear trend in injection systems, much less than 10% of the mass of solid samples, with an even our sample actually would make it onto distribution of sample sizes reported (5). the chromatographic column! However, the questions on sample size in this survey were separated by physical Concentration state so it is easier to observe trends. As in the past two surveys (4,5), there As shown in Table VI, 59% of survey was no change in the initial analyte respondents stated that they use solid concentration in the sample belonging samples of 1 g or less. Turning to larger to survey respondents. In each survey, samples, 17% report sample sizes of 53% of the samples had analyte 10 g or larger. The mode for sample size concentrations of >1 ppm and 47% appears to be about 0.5 g. encountered analyte concentrations of <1 ppm. While there were no apparent Final Volume of trends, Majors (5) discussed the need for Sample Before Injection extraction and concentration methods In most chromatographic methods, the like SPE and others that provide the pretreated sample must be in solution requisite trace enrichment and the use before injection onto the column. Often of sensitive detection systems. He also sample aliquots are retained for possible mentioned that analyte derivatization reanalysis. If any trend is gleaned from may provide improved detection. our survey, it is the shift away from small Given the potential for derivatization sample volumes. Table VII compares to improve the determination of trace survey results from 2002 (4) and 2013 (5) analytes, it is interesting to note that with 2015. For sample volumes <1 mL, the those that use derivatization decreased 2015 data revert back to that observed in from 49% in 2013 to 28% in 2015, see 2002, leading to a continued rise in the Figure 3. number of samples in the 1–2 mL range. Sample volumes greater than 10 mL also Automation saw a sharp increase to 23% of samples. With just two years between surveys, That 59% can be directly accommodated dramatic changes in survey results by standard 2-mL autosampler vials is not should not be expected. However, if we unexpected, though this is slightly down take another look at the discussion on from 2013 and more aligned with the sample load (see Table V), what follows 2002 results. Regardless, even if one were is perhaps the most dramatic result in

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Other C1 C2 Cyclohexyl Diol PSA C4 Florisil Amino Cyano Weak cation exchange Af nity Phenyl Application speci c or proprietary Weak anion exchange Strong anion exchange Strong cation exchange C8 Silica C18 0 10 20 30 40 50 60 70 80 Total respondents (%)

Figure 5: Silica-based SPE chemistries used, as reported in 2013 (blue) and 2015 (red).

the present survey. The number of survey throughput does not justify automation respondents using automated sample decreased from 55% in 2013 to 51%. Note preparation jumped significantly, from that 51% is only slightly more than the 29% in 2013 to 39% in 2015. This change 48% who have 50 or fewer samples per likely follows from what was reported instrument per week. At the same time, above. Those with more than 200 those that do not consider automated samples per instrument per week rose sample preparation necessary (31% from 8.5% to 14.5% and those expecting versus 27% in 2013) is roughly the same further increases in their sample load as the number that have fewer than 20 increased from 48% to 54% over the samples per instrument per week plus same survey periods. Perhaps to gain less than half of those with 21–50. In further understanding of this trend, we addition to the sharp increase in those should examine the rationale for those using automated sample preparation, the who have not adopted automated sample number planning to use these approaches preparation. Those that state their sample within next year is also up, from 4% to 7%,

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Other

Weak cation exchange

Application speci c or proprietary

Weak anion exchange

Strong cation exchange

Strong anion exchange

Neutral polymer

05 10 15 20 2530 35 40 45 50 Total respondents (%)

Figure 6: Silica-based SPE chemistries used, as reported in 2013 (blue) and 2015 (red).

and those reviewing its use is the same techniques, having been developed in the as in 2013, 11%. Cost as a reason against 1970s, while wide-spread developments the use of automated sample preparation of new technologies for the extraction of also grew, from 24% to 31%. solid samples such as SFE, accelerated Regarding the types of automation solvent extraction, and microwave- used, autosamplers still predominate and assisted extraction began around 1990. their use is unchanged from the 2013 SPE is used for both the extractive survey (83% of those using automation). isolation of analytes from liquid samples Full laboratory robotics was up, from and for the cleanup of post-extraction 7% to 14%, and use of automated liquid solutions. A number of techniques based handlers was down, from 17% to 9%, but on solute adsorption onto stationary there was no change in other types of phases, including SPME, stir-bar sorbent automated equipment. extraction, and dispersive SPE (like matrix solid-phase dispersion and QuEChERS), Solid-Phase Extraction came about at least partially as a result Each of the previous surveys outlined of the advantages of SPE as well as the strong use of SPE among survey needs for improvement. respondents. SPE is perhaps the oldest One of the advantages of SPE is the of the “modern” sample preparation variety of formats available including

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cartridges, disks, plates, and pipette Table VI: Solid sample size reported as a percentage of tips. The original SPE format was based survey respondents on syringe cartridges and this remains Sample Size Percentage of Respondents the most popular choice. Of survey <0.05 g 13.6 respondents who use SPE, 81% use 0.05–0.1 g 18.6 cartridges, down from 88% two years 0.1–0.5 g 18.6 0.5–1.0 g 8.5 ago. The overwhelming number of users 1.0–5.0 g 17.0 employ cartridges with 500 mg or less of 5.0–10.0 g 6.8 sorbent, but in 2015 this was down (65%) 10–50 g 10.2 from just over the historical trend of about >50 g 6.8 75% of users in 2013 and 2002. Most of this discrepancy can be attributed to 72% currently. It is interesting to note that those using the smallest amounts of SPE if we add the percent of SPE users that material (<50 mg). This decrease may employ each format (81% cartridges, 60% be explained by the use of other SPE disks, 60% plates, and 72% pipette tips), formats, as each of those reported saw that means each averages 2.7 of the four remarkable increase in utilization. The standard SPE formats in their laboratory. disk format has seen growth from use Turning to the types of phases in SPE, by 24% in the SPE community in 2002 77% of users claim to employ silica-based to 37% in 2013 and up to 60% in 2015. phases, 44% use polymeric phases, and The most popular disk size is 47 mm, so 31% use inorganic and other phases. the increased use is somewhat puzzling Since the advent of this survey, octadecyl since we reported earlier that drinking (C18), silica, octyl (C8), and ion-exchange and waste water analyses declined. The resins have dominated the phase increased analysis of biological fluids chemistry of silica-based material and that and tissues would explain the increased is true again in this survey, see Figure 5. use of the 96-well plate and pipette tip Our desire for analytical selectivity in the formats. The high-throughput well format, sample preparation process is reflected used for the rapid isolation of solutes in by the growth in the use of affinity and drug discovery and related scenarios, was application-specific or proprietary phases. introduced in 1996 (3) and in less than 20 In fact, the use of application-specific years has seen its growth expand to 14% phases rose from 6% to 15%. Other than of SPE users in 2002, 39% in 2013, and those phases just mentioned, each of 60% in 2015. There was no consensus the other phases were reported by no regarding the sorbent mass in the well- more than 10% of SPE users. Regarding plate format. Meanwhile in 2013, the polymeric phases, their use was down pipette tip approach was used by 52% of (44% compared with 62% in 2013). SPE practitioners, and that has grown to However, as observed in Figure 6, the

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Company–sponsored seminars Cartridge, disk, plate design SPE accessories available Brand name Application bibliography Compatibility with automation Solvent volume required Direct application support Cartridge, disk, plate material Range of chemistries Literature methods Free samples Validated protocols Cost Labor time Batch-to-batch reproducibility 0 10 20 3040 50 60 70 80 90 100 Total respondents (%)

Figure 7: Criteria rated as “very important” in selecting an SPE product according to the 2013 (blue) and 2015 (red) surveys.

overall trend in the specific phases used is important, and not important) of the similar. Finally, of those that use inorganic criteria in selecting SPE products, batch- phases, the use of Florisil and alumina to-batch reproducibility reigned again, dropped strongly, from 58% in 2013 to with a similar response to 2013 (Figure 7). 47% currently for Florisil and 54% to 27% Direct application support, free samples, for alumina. This is offset somewhat by and available formats (cartridge, disk, a sharp increase in the application of plate material) each greatly gained in graphitized carbon black, increasing from relative importance. Other criteria that 40% to 60% over the past two years. gained in importance included labor time, Graphitized carbon, with a positively cost, validated protocols, compatibility charged surface has both reversed-phase with automation, and brand name. and anion-exchange properties and it can Looking at the criteria related to methods retain analytes covering a broad range of and support, including brand name, polarities. makes one wonder whether SPE users When rating the relative importance are increasingly accepting of methods (very important, somewhat important, developed by others or if there are

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Table VII: Final sample volume after sample pretreatment increased concern with time and labor and before chromatographic injection, reported as a per- centage of survey respondents for 2002, 2013, and 2015 intensity and interpretation of results, Final Sample Volume 2002 2013 2015 less concern with sample recovery and <1 mL 25 38 26 contamination, and similar concerns with 1–2 mL 27 31 33 lack of reproducibility and insufficient 3–10 mL 29 14 19 knowledge of the sample. It would >10 mL 19 17 23 be easy to flippantly suggest that interpretation of results and insufficient currently a growing number of novice knowledge about the sample are more workers seeking guidance. Alternatively, reflective of the chemist than the sample if we peruse the data for the criteria preparation, but when looking at the deemed not important, not unexpectedly, “other” responses comments on limited those rated very important (batch-to- staffing and insufficient training, one batch reproducibility, labor time, and might place the concerns systemically cost) were also the least frequently rated rather than on the individuals in the as not important. These three criteria, laboratory. Another problem encountered along with range of chemistries, are in sample preparation is concern the only ones rated not important by about handling limited sample sizes. less than 10% of SPE users. Conversely, On a positive note, in the 1996 survey cartridge, disk, and plate design, (3), poor sample recovery and lack of brand name, application bibliography, reproducibility were noted as concerns by compatibility with automation, and more than 35% of respondents and these direct application support all were rated have slipped to 21% for sample recovery as not important by more than 30% of and 22% for irreproducibility, respectively. respondents. This decrease is most likely because of the increased attention and advances Top Problems Encountered in extraction and sample preparation in Sample Preparation technologies over the past generation. When survey respondents were asked Kudos to those researchers and vendors to identify the top problems they involved in this trend! encounter in sample preparation, they sang the same tune as in previous Emerging Sample surveys. Time, recovery, reproducibility, Preparation Techniques cost, and interpretation of results have We previously noted that of the sample been reported since the question preparation techniques listed in Figure was first asked in 1996 (3). The 2013 3, respondents expressed an expected and 2015 survey results are compared increase in derivatization, QuEChERS, graphically in Figure 8. We can see SPME, accelerated solvent extraction,

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Other

Contamination

Interpretation of results

Poor sample recovery

Insuf cient knowledge about sample

Lack of reproducibility

Cost

Time, labor intensity

0 10 20 30 40 50 60 70 80 Total respondents (%)

Figure 8: Top problems encountered in sample preparation in 2013 (blue) and 2015 (red).

evaporation, homogenization, internal general use. Techniques joining that list standard addition, large-volume in the most recent survey are described trace enrichment, matrix solid-phase below. dispersion, microwave-assisted extraction, solvent exchange, SFE, and ultrasound- Stir-Bar Sorptive Extraction assisted extraction. We separately asked Already used by 5.6% of this survey a free-form question to name the three respondents, stir-bar sorptive extraction sample preparation techniques on the uses a stationary phase coated onto horizon that could become commonplace a magnetic stir bar for the removal of in the next five years. Techniques analytes from liquid solution. Because mentioned that are considered carry- of the large volume of stationary overs from 2013 are accelerated solvent phase (due to the large surface extraction, QuEChERS, SPME, supported area), the sample capacity is high. liquid extraction, microwave-assisted Correspondingly, the adsorption and extraction, and room-temperature ionic desorption times are long, but this liquids. In the summary of the 2013 survey potential drawback is negated by the (5), Majors provided a brief description of availability of automation. Although the broad applications of these and their the technique holds promise, stir-bar

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sorptive extraction is limited in the Conclusions number of stationary phases available Although sample preparation is still and vendors promoting the technique. considered a time- and labor-intensive step in an analytical scheme, its Restricted-Access Media importance is undeniable. We have LCGC first reported the use of these looked at short-term trends in sample materials as liquid chromatography (LC) preparation over the past two years, stationary phases in 1997 (6). Just as the especially in the use of automation and use of other application-specific SPE newer instrumental extraction techniques. phases are seeing increased use, it follows Similarly, some long-term trends during that the tremendous selectivity advantages the past 10–25 years of this survey have of restricted-access media is of strong been noted. interest for high-volume analyses. Acknowledgment Direct Analysis in Real-Time MS I would like to thank those readers who When introduced about a decade ago, took the time to respond to the rather this approach to sample introduction for lengthy survey. The information provided MS seemed like the panacea—no sample helps to keep all readers up on the latest preparation at all. Gas, liquid, and solid technology in sample preparation. samples can be ionized under ambient conditions for MS analysis. After 10 years, References (1) R.E. Majors, LCGC 9(1), 16–20 (1991). the growing pains are being worked out (2) R.E. Majors, LCGC 10(12), 912–918 (1992). and the promise of the technique may (3) R.E. Majors, LCGC 14(9), 754–766 (1996). (4) R.E. Majors, LCGC North Am. 20(12), 1098–1113 (2002). soon be fully realized. (5) R.E. Majors, LCGC North Am. 31(3), 190–202 (2013). (6) K.-S. Boos and A. Rudolphi, LCGC 15(7), 606–611 (1997). Matrix Solid-Phase Dispersion Especially useful for tissue samples, This article originally appeared in LCGC this dispersive SPE approach is seeing North America, 34 (3), 174–188 (2016). a resurgence because of the similarity with the initial portion of the QuEChERS method. While initially developed for Douglas Raynie the isolation of pesticides in fruits and “Sample Prep Perspectives” editor Douglas E. Raynie vegetables, QuEChERS has been refined is an Associate Research Professor at South Dakota State University. His research interests include green as a screening tool for a wide variety of chemistry, alternative solvents, sample preparation, high resolution chromatography, and bioprocessing applications. As the myriad applications in supercritical fluids. He earned his PhD in 1990 at are sorted out, the matrix solid-phase Brigham Young University under the direction of Mil- ton L. Lee. He is also a member of LCGC’s editorial dispersion technique should settle into its advisory board. comfort zone.

28 | July 2017 | LCGC Stop searching for filters Trust Whatman™ GE

gelifesciences.com/hplc GE, GE monogram, and Whatman are trademarks of General Electric Company. © 2017 General Electric Company. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden For local office contact information, visit gelifesciences.com/contact. 29273662 AA 06/2017 30 | July 2017 |July 30 |LCGC Click here here Click the video the P Douglas E. Raynie and Ronald E. Majors E Th P to watch watch to reparat o xplor Workflow QC Optimizing Lab ro ssi S ponsored u b g

i l i ng t ng h i t p i i on e u we address the analytical need forwe speed? address the analytical adequately address the issue of time? How can preparation? Do modern methods extraction considerations, what high-throughput is sample toward separations. on faster these Based chromatography moving advances are also techniques addressextraction modern time, on sample preparation. newly developed While spend time easily amajority of analysis the total preparation procedures can and that analysts the most frequent problem for area sample have shownSurveys typically is that time procedures. the bottleneck analytical as in Sample preparation been often viewed has time, along with cost and solvent use, is is use, solvent and cost with along time, that shown consistently have (1–3) surveys because preparation sample analytical to admonition Stones’ Rolling the apply to easy It’s want.” you what get always can’t “You sang, famously band roll and rock greatest world’s self-proclaimed The s t Sample Sample t h o e f H i g h -

getty Images/xxxxxxxx Sample Prep Sample Prep High-Throughput Filtration Overview Trends Sample Prep

among the most significant desires Wells (4) presented a list of some of analysts. Traditional sample of the uses of filtration microplates in preparation methods are often the pharmaceutical development, including: rate-limiting step in the overall sample • clarification of acid, base, or organic analysis process. We can envision digests of plant materials in medicinal three approaches for addressing chemistry, the desire for high-throughput • SPE of natural products, sample preparation: parallel sample • solution-phase synthesis in processing, automation, and combinational chemistry using resin improvements in the process kinetics. scavenger media, • dye terminator removal, Evolution of High-Throughput • plasmid DNA binding, Analysis from Microplates • lysate clarification, Over the past several years, we’ve heard • membrane-based proteolytic digestion increasing calls for high-throughput before MALDI-MS, sample preparation. But what is high- • filtration of precipitated proteins, throughput sample preparation? In • filtration of plasma or serum samples many cases, high-throughput stems in combination with direct injection from the high-throughput screening techniques, approach to combinatorial chemistry. • filtration of reconstituted extracts, and This approach is the most established, • solid-supported LLE, SPE, or exclusion based on the 96-well plate format. These chromatography. microplates have been established for As we can see, the development of 96- two to three decades. Key considerations well plates and the major applications of for the acceptance of this approach this approach are in bioanalysis of liquid are standardization of microplate samples. This is because the transfer of dimensions and attributes by the liquid samples from analytical operation Society for Biomolecular Screening and to another is somewhat straightforward. development of ancillary devices like Recently, Scoffin (5) reviewed liquid repeating pipettes, vacuum manifolds, transfer in high-throughput systems. and more. Countless vendors are These liquid-handling systems operate involved. Sample preparation and analysis either by air displacement or positive using microplates includes liquid–liquid displacement and can reliably work with extraction (LLE), protein precipitation, microliter volumes of volatile or viscous solid-phase extraction (SPE), fluorescence, liquids. Other combinations of liquid matrix-assisted laser desorption– handling and sorbent-based extraction, ionization mass spectrometry (MALDI- including solid-phase microextraction MS), and separations. (SPME), automated disposable pipette

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Figure 1: Example of turbulent flow profile.

extraction, and microextraction by sample preparation purposes, facilitates packed sorbent have also been used in radial diffusion (that is, mass transfer) high-throughput approaches (6,7). By to the stationary phase. Two years ago, combining these solvent-free, sorbent- sample preparation applications of TFC based extractions with syringe-needle or were discussed in this column (8). To pipette configurations, direct extraction achieve turbulent flow conditions, large, and liquid transfer is accommodated. porous particles are combined with high flow rates. In general, 30–60 μm particles Turbulent Flow Chromatography packed in 0.5–1.0 mm i.d. columns with in Sample Preparation flow rates greater than 1 mL/min and up In recent years, turbulent flow to 6 mL/min achieve these conditions. chromatography (TFC) has emerged as Dramatic reductions in time are observed, a high-speed extraction alternative for while maintaining accuracy, precision, liquid samples. When flow rates increase detection limits, and other figures of significantly, the flow pattern shifts merit. Low-molecular-weight compounds from a laminar, bullet-shaped profile to readily diffuse into the porous particles, a turbulent profile. An example of this whereas larger compounds are less flat, turbulent flow profile is shown in retained and are eluted to waste. Proteins Figure 1. Turbulent flow minimizes zone are reported to be more selectively spreading and, most importantly for removed from biosamples using TFC than

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100 TFC

90

80 RAM 70 SPE

60

50

Percent excluded 40

30

20

10

0 5000 15,000 25,000 35,000 45,000 55,000 65,000 75,000 MW (Da)

Figure 2: Comparison of turbulent flow chromatography (TFC), restricted-access media (RAM), and solid-phase extraction (SPE) cleanup columns as a function of the molecular weight excluded during sample cleanup. Adapted from reference 9.

restricted-access media (RAM) or solid- consumption. Observed matrix effects phase extraction, as illustrated in Figure require the use of standard addition. 2 (9). Thus, small molecule analytes in Recent reviews (10–13) have illustrated samples like whole blood, serum, plasma, the use of TFC for biofluids, foods, and or urine can be delivered to the injection environmental samples. port when coupled to high performance liquid chromatography (HPLC) in an on- What About Solid Samples? line manner using column switching. So far, we’ve focused the discussion of Although minimal sample handling is high-throughput sample preparation on necessary, resulting in fast analysis, the liquid samples. The reasons are two-fold, high flow rates necessary to achieve as previously mentioned. Liquid handling turbulent flow result in a large solvent is easier than manipulation of solids

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Table I: Average diffusion coefficients obtained from takes 20 min, while several-fold faster the extraction of oils from ground, roast coffee than Soxhlet or shake-flask methods, is Average Diffusion Extraction Technique Coefficient (cm2/s) still slow compared with 5-min analyses.

Soxhlet extraction 9 × 10-10 In the days of 6–48-h Soxhlet extraction

Automated solvent extraction 8 × 10-9 and 30–60-min chromatographic analysis,

Ultrasound extraction 2 × 10-8 the ratio of sample preparation time to

Pressurized liquid extraction 4 × 10-8 chromatography run time was 6–96:1.

Microwave-assisted extraction 6 × 10-8 However, it is common to perform, for example, 12 Soxhlet extractions and the microplate technology drove simultaneously, so the ratio becomes the development of high-throughput 0.5–8:1. SFE, PLE, and MAE extractions methods. How can we make the analysis commonly take 20 min, but have we really of solid samples high-throughput? Do gained anything? It is now routine to the more recently developed extraction perform chromatography with 10–30-min techniques take us in that direction? run times or faster. So the ratio remains During the time when 30–60 min 0.5–6:1. chromatography runs were standard, The fundamental difference between Soxhlet extractions took 6–48 h. This extracting solid samples and liquid bottleneck was even further exacerbated samples is the role of diffusion. Diffusion when spectroscopy or other rapid analytical in liquids at room temperature is on the procedures were used. During the past 20 order of 10-5 cm2/s, while diffusion in years or so, several automated techniques solids at the same temperature is on the (including supercritical fluid extraction order of 10-9 cm2/s. But what about those [SFE], microwave-assisted extraction solid extraction methods that operate [MAE], and pressured liquid extraction at elevated temperatures? We have [PLE]) have been developed, which provide conducted a systematic comparison of the hope for high-throughput sample SFE, MAE, PLE, and sonication extractions analysis. In addition to automation of the to examine the rate-limiting role of extraction procedure, these methods are diffusion in these extraction methods. characterized by very rapid extraction We extracted the oils from ground, times compared with the traditional roast coffee using these methods and methods. However, simultaneously to determined the diffusion coefficients using the development of these techniques, the hot-ball model (14). Extractions were advances in sensors, mass spectrometry, performed using dichloromethane at 100 and chromatography have rendered even °C under suboptimal conditions, which these modern technologies to remain would allow the determination of the the rate-limiting part of an analytical diffusivity. Soxhlet extraction is performed procedure. Sample preparation that at some elevated temperature near, but

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100

80

60 Soxhlet Automated solvent extraction PLE 40 MAE Ultrasound 20

0 0 10 20 30 40 50 60

Figure 3: Relative percent of oil extracted from 725-μm coffee particles as a function of time (min) using various extraction processes. Adapted from reference 15.

below, the boiling point of are obtained. The results show that analyte 39.8 °C, while the modified Soxhlet diffusion values, under similar extraction approach includes leaching in boiling conditions applied across each method, solvent as the main portion of the were within the same order of magnitude. extraction. Ultrasonic extractions are It seems unlikely that temperatures greater performed in open-vessels, so the bulk than those used in these techniques solvent cannot exceed the boiling point, will be routinely applied for analytical but on a molecular level temperatures extractions. Thus, when developing during cavitation can reach 5000 K. PLE high-throughput extraction systems, the and MAE in this work were performed kinetics of extraction will not differentiate at 100 °C. Figure 3 displays the kinetic these alternative extraction procedures. curves from these extractions (15). When PLE, MAE, and SFE seem to be the the hot-ball model is applied to these data, preferred approaches for developing high- the diffusion coefficients shown inTable I throughput processing, but advantages

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with each technique will arise because of mass spectrometry eliminate the need instrumental configurations. for sample preparation entirely, which is perhaps the fastest method of all. Is High-Throughput Sample Preparation Necessary? References (1) R.E. Majors, LCGC North Am. 9(1), 16–20 (1991). As discussed, the major approaches (2) R.E. Majors, LCGC North Am. 20(12), 1098–1113 (2002). to achieve high-throughput sample (3) R.E. Majors, LCGC North Am. 31(3), 190–202 (2013). (4) D.A. Wells, High Throughput Sample Preparation Methods preparation stem from parallel sample and Automation Strategies (Elsevier, Amsterdam, The Neth- processing, automation, and the kinetics erlands, 2003), pp. 199–254. (5) K. Scoffin, Am. Lab. 46, 17–19 (2014). of the sample processing procedure. The (6) J. Pereira, C.L. Silva, R. Perestrelo, J. Goncalves, V. Alves, and J.S. Camara, Anal. Bioanal. Chem. 406, 2101–2122 (2014). success observed in the high-throughput (7) F. Mousavi and J. Pawliszyn, Anal. Chim. Acta. 803, 66–74 sample preparation of liquids comes, (2013). (8) J.L. Herman, T. Edge, and R.E. Majors, LCGC North Am. in part, from automation and parallel 30(3), 200–214 (2012). processing. The microplates process 96 (9) O. Nunez, H. Gallart-Ayala, C.P.B. Martins, and P. Lucci, J. Chromatogr. A. 1228, 298–232 (2012). or more samples simultaneously. The (10) J. Pan, C. Zhang, Z. Zhang, and G. Li, Anal. Chim. Acta 815, 1–15 (2014). instrumental approaches to extracting (11) O. Nunez, H. Gallart-Ayala, C.P.B. Martins, P. Lucci, and R. solids are also configured for automation Busquets, J. Chromatogr. B. 927, 3–21 (2013). (12) M.D. Marazuela and S. Bogialli, Anal. Chim. Acta. 645, 5–17 and parallel processing. The kinetics of (2009). extraction is fundamentally limited by (13) L. Couchman, Biomed. Chromatogr. 26, 892–905 (2012). (14) K.D. Bartle, A.A. Clifford, S.B. Hawthorne, J.J. Langenfeld, practical considerations. So perhaps the D.J. Miller, and R. Robinson, J. Supercrit. Fluid. 3, 143–149 (1990). best approaches for high-throughput (15) J.L. Driver and D.E. Raynie, in preparation (2014). analysis will result from the integration of methods. Just as turbulent flow This article originally appeared in LCGC chromatography is coupled to liquid North America, 32 (6), 396–403 (2014). chromatography (LC), thermal desorption is coupled with gas chromatography (GC), Douglas Raynie and 96-well microplates are coupled is an Associate Research Professor at South Dakota State University. His research interests include green to MALDI-MS, similar methodologies chemistry, alternative solvents, sample preparation, combining rapid sample and processing high resolution chromatography, and bioprocessing in supercritical fluids. He earned his PhD in 1990 at parallel approaches will begin to address Brigham Young University under the direction of Mil- ton L. Lee. He is also a member of LCGC’s editorial this need. However, the analyst must advisory board. also keep in mind what is truly desired.

High-throughput sample preparation is Ronald E. Majors not the ultimate goal, but rather high- “Sample Prep Perspectives” Editor Ronald E. Majors is an analytical consultant and is a throughput analysis. Chemometrics and member of LCGC’s editorial advisory board. other statistical approaches can glean the Direct correspondence about this column to [email protected]. desired information from minimal analysis, and dilute-and-shoot or direct analysis

36 | July 2017 | LCGC 37 | July 201737 |July |LCGC the article the Click here here Click here Click T C F F Dwight Stoll Dwight T C F F the filter filter the selector to view view to to read read to o Do i i o Do i i h h ltrat lter ltrat lter Whatman Filter Selector Whatman Variation toStandardize Reduce romatograp romatograp SPONSORED SPONSORED s an s an i i on i on i d d chromatography (HPLC) John instruments. operation of liquid high performance and filtering theyas relate proper,to reliable not with the accuracy we would like. To my amazement, it still pumped liquid, albeit the pump head sits was literally packed with dirt. full of dust and the front area of the pump where a Minnesota winter—the electronic boards were it had sat out in the middle of a farm field through pump from a laboratory in Kansas that looked like and weekends. I vividly recall receiving an LC school days was doing instrument repair on nights balanced the checkbook during my graduate more-delicate components. One of the ways I practices designed to protect the instrument’s spite of our occasional justified neglect of best chromatography (LC) works remarkably well in Sometimes, the instrumentation we use in liquid results. quality methods and high “clean” mobile for important phaseis robust column in Delivering samples to the analytical n Li n Li In this article, Idecided to focus onfilters q q h h uid uid y— y— Wh Wh at at

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Dolan has written extensively and often The topic of filters and filtering is very on these topics in the past (1–4), and important in LC because a number yet many of the problems I discuss with of components in a LC system are LC practitioners in the field—and with susceptible to premature failure if students in my own laboratory—are in particulates are present in the mobile- some way related to filters and filtering. phase flow path. For example, dust or Technologies, practices, and the demands other particulates in the mobile phase of our laboratory settings change, pulled from a solvent bottle can cause too, and so I’d like to discuss what has pump check valves to partially fail (they stayed the same and what has changed, become leaky) or entirely (the pump while providing some guidance for our produces no flow at all). Perhaps the most everyday work. obvious failure mode involves particulates In preparation for writing this piece, from injected samples or instrument I informally surveyed several people, components accumulating at the column because I was interested to know inlet, resulting in a plugged column. what folks are actually doing in their In the following sections, I will discuss laboratories. I consider all of these some of the most important points in an people experts in LC, with an average of instrument setup and method related to about 15 years of experience, and they filters and filtration. In each case, I will represent a broad cross-section of our describe best practices from the point community, ranging from pharmaceutical of view of the instrument and will also and chemical companies to instrument discuss cases in which this advice may not manufacturers. What was strikingly be strictly followed for good reason. We evident from this survey was that people will see that many of these considerations handle the topic of filters and filtering in boil down to thinking about risk and diverse ways, even though instrument benefit in terms of the potential benefit manuals and books on LC may suggest derived from deviating from best only one way. Our reality today is that practices and the risks associated with the practice of LC is strongly influenced doing so. by a number of drivers that the manuals don’t consider, including minimizing Filtering to Protect the Pump the number of laboratory operations to From the point of view of an LC pump, minimize variation in analytical results, ideal solvents are absolutely free of and optimizing the costs of analytical particulate matter. Particulates can results from a holistic point of view (in interfere with the sealing interface other words, analyzing the benefit of between the check valve ball and seat, component X to the method relative to causing it to leak or to fail entirely, the cost of the component). resulting in inconsistent or no flow

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from the pump. Particulates can also available for HPLC, including phosphate accelerate wear on pump pistons and buffers, buffers with ion-pairing reagents seals, which can result in leaks and flow- added, and so on. The convenience rate inaccuracy. These problems usually associated with using these premixed manifest themselves as noisy or irregular solvents comes at a significant increase baselines, unexpected fluctuations in in purchase price over pure solvents, the operating pressure reported by the however, so users must decide if the pump, and variations in retention time. increased cost is worth the benefits they Most sources of advice on this topic offer. Some laboratories find that the recommend that solvents should be increased cost of premixed solvents is filtered to 0.45 µm before use. Modern offset by savings in labor and reduced LC pumps come equipped with filters that downtime that would otherwise result are fitted to the inlet end of solvent lines from errors in mobile-phase preparation. that pull solvents from their reservoirs, but these filters have much larger porosity Mobile-Phase Additives and are not designed to retain particles The majority of LC methods in use today at the 0.5-µm level. Off-line filtration, involve some type of mobile-phase however, adds cost (both consumables additive, including buffering agents and and operator time) and is a potential ion-pairing reagents. If we choose not source of both chemical and particulate to use the type of premade solvents (yes, it happens) contamination of the described above, then we have to make solvent. Reputable manufacturers of a choice: Do we filter the solvent after solvents sold specifically for use in adding the additives, or not? At this LC filter their solvents at the point of point there is an opportunity to divide packaging (typically to 0.2 µm, and some the question over two scenarios—those to 0.1 µm), so one way to avoid in-house that involve liquid additives (for example, filtering altogether is to purchase and formic acid, ammonium hydroxide, and use premixed solvents. There was a phosphoric acid), and those that involve time when choices for these solvents solid additives (for example, potassium were limited to water and neat organic phosphate, sodium perchlorate, and solvents, but these days most commonly sodium chloride). Among the experts I used mobile phases can be purchased surveyed, very few reported that their from major manufacturers as premixed laboratories filter solvents involving only solutions. For example, 0.1% formic acid liquid additives. I would report the same in water, 0.1% formic acid in acetonitrile, for my laboratory. The responses from and 0.1% ammonium acetate in water the experts about solvents involving solid are all readily available. There is also additives are mixed. A majority, but not an incredible array of premade buffers all, of their laboratories routinely filter

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solvents containing solid additives. In my • Risk of introducing chemical and laboratory we often base our decision biological contaminants during on the amount of solid additive used. If filtering:These days, very high-quality the amount of additive is small and it is membrane filters are available for highly soluble in the solvent—say filtering LC solvents that are unlikely to 10 mM ammonium acetate in water—we contaminate the solvent themselves. don’t filter, but we always filter solutions But, the filter is only one component containing high concentrations of of the filtration process. The rest additives. The compelling reasons to filter of the filtration apparatus must be in these cases include increased risk of cleaned properly to avoid carryover of particulate contaminants present in the particulate and chemical contaminants solid additive, particulates present due to the filtered solvent. This approach to incomplete dissolution of the additive, is most certainly possible, but it is and, in the case of buffer–organic solvent dependent on well trained operators, mixtures, precipitation of the additive standard operating procedures, and upon addition of the organic solvent. a laboratory culture of respect for cleanliness. This issue of chemical and Why or Why Not? biological contaminants is especially In the face of these compelling reasons important for methods focused on to filter, especially when solid additives trace-level determinations, because the are used, one might reasonably ask—why contaminants will be observed as noise not? Here is a short list of reasons, the or interferents when using very sensitive significance of which often depends on detectors (5,6). context. • Fewer steps equal less opportunity for mistakes: One of the themes that • Cost of filtering versus costs resulting emerged from my survey of experts from pump problems: If the risk of a is that there is a continuous drive for solvent-related problem associated with simplification of analytical procedures. the pump can be minimized through If a step can be eliminated without use of liquid additives and high-quality compromising the quality of the reagents, the cost associated with in- analytical result, it is because fewer house filtering may outweigh costs steps tend to translate into lower associated with pump down-time. On variability of results. the other hand, if your laboratory is a 24/7 operation and pump robustness is Microbial Contamination critical, then you may choose to prioritize Finally, the topic of microbial growth in-house solvent filtering to maximize in solvent reservoirs is important. If instrument up-time. microbes begin growing in the solvent

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container feeding the pump, there the “right amount” is case specific) of is potential for the instrument to be organic solvent such as acetonitrile to contaminated both physically and the buffer for this purpose. chemically. Here, too, some themes emerged from the experts I surveyed. Filtering to Protect the Column The following best practices will help The size of the interstitial spaces between minimize problems with microbial growth, particles of packed columns typically used which is limited to aqueous solutions: in LC is a small fraction of the diameter • Resist the temptation to top off solvent of the particles themselves. Thinking of containers by adding new buffer to it this way, it becomes evident that the old buffer. Topping off provides a packed bed itself can act as an effective path for cross-contamination of buffer filter; clearly we want to employ the batches—even if the new buffer is column for separation, not as a filter. The perfectly sterile, it will be contaminated frits on the ends of the columns serve two immediately if added to a solvent purposes then: They retain the particles reservoir containing old buffer. Avoiding inside the column, and they prevent this practice adds some waste when the particulates carried by the mobile phase last drops of old buffer are thrown away, from entering and partially blocking flow but this minor waste is a small price to through the particle bed. Accumulation pay for avoiding cross-contamination. of particulates on the frit at the inlet side • For buffered aqueous solutions, of the column can also lead to plugging particularly those near neutral pH, of the frit, rendering the column unusable prepare concentrated stock buffer because of the increased pressure drop solutions (such as a solution containing over the frit. In the preceding section I a 10× concentration of buffering agent), discussed the mobile phase as it is drawn refrigerate them upon storage, and into the pump as a source of particulates. dilute small quantities for use when But even if the mobile phase entering needed. Prepare only enough of the the pump is perfectly particulate-free, working buffer to last one or two days there are several potential sources of to limit the time available for microbes particulates entering the flow stream to grow at room temperature. between the pump and the column. Most • Add some antimicrobial agent to prominent among these are shedding of the buffer. Historically, sodium azide pump-seal material (chunks of polymer); was used for this purpose at low shedding of valve rotor seals (again, concentration (~0.05%), but this practice chunks of polymer), including the sample is becoming less common and many injection valve and column switching laboratories are choosing to simply add valves; and the sample itself. The topic a small amount (many labs use 10%, but of particulates in the sample is one

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that clearly crosses over into the area laboratories have an in-line filter installed of sample preparation, and one could ahead of the analytical column, which is easily write an entire column on this topic very surprising to me. Replacement filters alone. Here, we will focus on the first two cost anywhere from a few dollars to $25 potential sources, the instrument-related depending on their features, but it is easy sources of particulates. to justify replacing a few of these if we can significantly increase the lifetimes In-Line Filters Versus Guard Columns of our analytical columns and improve In the context of a LC instrument, an instrument robustness in general. It is in-line filter is usually one that is placed important to note that some applications in the mobile-phase flow path at some involving very small peak volumes (for point between the pump outlet and example, small particle sizes and short the column inlet. These filters have columns) will be more susceptible to evolved over the years to the point that peak broadening inside the in-line filter. the most high-performing ones can be In these cases one has to think carefully used at very high pressures, have small about which is more important— throughpore sizes (down to 0.2 µm), and minimizing dispersion, or protecting the have internal volumes in the microliter column from particulates. range so as to limit contributions to Guard columns are essentially very peak broadening. The sole purpose short versions of the analytical column of these filters is to collect particulates (often with the same packing material in the mobile phase as a means of and diameter, but lengths on the order preventing damage to downstream of 5 or 10 mm) that are screwed into the components from these particulates. In column inlet. One can argue that guard my laboratory we do not regularly use columns can also serve as particulate in-line filters immediately after the pump, filters to protect the analytical column, but we are very disciplined about using but this approach is a much more them between valves with polymeric expensive way of doing so (on the order rotor seals (for example, autosampler of 10–20% of the column purchase and column-switching valves) and the price per guard column). Instead, guard analytical column. We find that these columns are more appropriately used seals shed a lot of particles as they wear, for chemical protection of the analytical and that capturing them with an in-line column. For example, sample constituents filter generally increases the lifetimes that are very highly retained by the of our analytical columns and small analytical column, or perhaps will not diameter capillary connection tubing. I even be eluted under the conditions of would say that only about 50% of the LC the experiment, can be trapped by the instruments I see in a year in different guard column. The contaminated guard

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column can then be replaced with a new (1)References J.W. Dolan, LCGC North Am. 28, 28–30 (2010). one after the analysis of some number of (2) J.W. Dolan, LCGC North Am. 26, 352–355 (2008). (3) J.W. Dolan, LCGC North Am. 3, 956–960 (1985). samples at a fraction of the price of a new (4) J.W. Dolan and V.N. Berry, LCGC N. Am. 1, 542–544 (1983). analytical column. The experts I surveyed (5) B.O. Keller, J. Sui, A.B. Young, and R.M. Whittal, Anal. Chim. Acta. 627, 71–81 (2008). reported more frequent use of in-line doi:10.1016/j.aca.2008.04.043. filters than guard columns, citing the cost (6) S. Williams, J. Chromatogr. A. 1052, 1–11 (2004) 1–11. doi:10.1016/j.chroma.2004.07.110. of guard columns as being generally too high to justify their use in a uniform way. This article originally appeared in LCGC Closing Thoughts North America, 35 (2), 98–103 (2017). In this article, I made generalizations where possible about when and why to filter. Of course there are special Dwight Stoll is Associate Professor and Co-Chair of Chemistry at situations where these generalizations are Gustavus Adolphus College in St. Peter, Minnesota. not applicable. For example, certain types He has authored or coauthored 48 peer-reviewed publications in separation science and more than of detectors, such as light-scattering 90 conference presentations. His primary research focus is on the development of two-dimensional liq- detectors used for polymer analysis, are uid chromatography (2D-LC) for both targeted and very sensitive to particulates in the mobile untargeted analyses. He has made contributions on the topics of stationary-phase characterization, new 2D-LC methodologies phase and may require more-thorough and instrumentation, and fundamental aspects including reequilibration in gradient elution reversed-phase LC and analyte focusing. He is the 2009 and more-consistent filtering of mobile- recipient of the John B. Phillips Award for contributions to multidimen- phase solvents. LC users should think sional gas chromatography, the 2011 recipient of LCGC’s Emerging Leader in Chromatography Award, and the 2015 recipient of the American Chemi- carefully about special requirements cal Society Division of Analytical Chemistry Award for Young Investigators in Separation Science. He is also a member of LCGC’s editorial advisory associated with their particular analyses board. Direct correspondence about this column via e-mail to LCGCedit@ and adjust their approaches to the use of ubm.com. filters and filtering accordingly.

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