Anal. Chem. 2010, 82, 4895–4910
Planar Chromatography Joseph Sherma Department of Chemistry, Lafayette College, Easton, Pennsylvania 18042 Review Contents History, Student Experiments, Books, and Reviews Theory and Fundamental Studies Chromatographic Systems Stationary Phases Mobile Phases Apparatus and Techniques Sample Preparation Thin Layer Chromatography Detection and Identification of Separated Zones Chemical Detection Biological Detection Thin Layer Chromatography/Mass Spectrometry Thin Layer Chromatography Coupled with Other Spectrometric Methods Sample Identification Software Quantitative Analysis Techniques and Instruments Applications Preparative Layer Chromatography Thin Layer Radiochromatography Literature Cited
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This review covers the literature of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) found by computer-assisted searching in Chemical Abstracts and the ISI Web of Science from November 1, 2007 to November 1, 2009. The literature search was augmented by consulting Analytical Abstracts, and the following journals that regularly publish papers on TLC were searched directly: Journal of Chromatography (parts A and B), Journal of Chromatographic Science, Chromatographia, Analytical Chemistry, Journal of Liquid Chromatography & Related Technologies, Journal of AOAC International, Journal of Planar ChromatographysModern TLC, Acta Chromatographica, and Acta Universitatis Cibiensis, Seria F, Chemia. Chemical Abstracts (SciFinder Scholar) cited 2583 references containing the search phrase “thin layer chromatography” during this review period, compared to 2290 citations for the last review period, November 1, 2005 to November 1, 2007; the actual totals are higher than these numbers because all worldwide journals publishing papers on TLC are not surveyed by this service, and all papers abstracted by it may not be recovered using only this one keyword phrase. No papers reporting new research on paper chromatography, the other main classification of planar chromatography, were considered to be important enough to be included in this review. Coverage is limited to significant papers representative of the current practice and important advances in the field of TLC, with specific sections on history and literature, fundamental studies, methodology, equipment, and instrumentation. TLC continues to feature a broad range of applications, such as analysis of pharmaceuticals, herbal medicines and dietary supplements, 10.1021/ac902643v 2010 American Chemical Society Published on Web 01/07/2010
biological and clinical samples, foods and beverages, environmental pollutants, and chemicals, and important new applications to specific analytes and sample matrixes of many types are cited in all sections throughout this review, especially in situ densitometric methods in Quantitative Analysis. The presentations at the International Symposium on HPTLC 2008, held in Helsinki, Finland, in June, 2008, and the papers contained in five special journal issues or sections provide an upto-date picture of the some of the most important technique and application areas in TLC research over the past 2 years. Three workshops at the Symposium covered HPTLC fundamentals, plant analysis, and validation criteria. The topics of the 23 lectures were divided into “News, Fundamentals, and Theoretical Aspects”, “Strong Features of HPTLC”, “Specific Applications”, and “Detection, Bioactivity Tests, and Coupling Techniques. The 33 posters were in five categories: “General”, “Pharmaceutical and Biomedical Analysis”, “Environmental and Forensic Analysis”, “Herbal Analysis”, and “Food Analysis”. The next HPTLC International Symposium will be held in Basel, Switzerland, July 6-8, 2011; details on attendance and presenting a poster or paper can be obtained at www.hptlc.com. Subjects of papers in a special issue on TLC of the Journal of Liquid Chromatography and Related Technologies [2008, 31 (13)], guest edited by Sherma and Fried, were HPTLC separation of phospholipids; HPTLC-densitometry determination of the neutral lipid content of the feces of BALB/c mice infected with the intestinal trematode Echinostoma caproni and in organs of the lizard Uta stansburiana; HPTLC-densitometry analysis of hydrochlorothiazide, walsartan, kandesartan, and enalapril in complex hypotensive drugs; TLC-direct bioautography as an alternative to high-performance column liquid chromatography (HPLC) for determination of cefacetril in cow’s milk; analysis of 11 antidepressive drugs by HPTLC-densitometry with a diode array detector (DAD) scanner; stirbar sorptive extraction-HPTLCfluorescence densitometry (SBSE-HPTLC-FLD) method for quantification of six polycyclic aromatic hydrocarbons (PAHs) in drinking and environmental water samples; application of densitometry to evaluate the visualizing effects of salicylanilide using Brilliant Green; quantitative silver ion TLC of triacylglycerols from sunflower oils; quantification of urethane in spirits; analysis of vitamin B12 in foods by TLC-bioautography; TLC investigation of the oscillatory transenantiomerization of L-alpha-phenylalanine and L-tyrosine; and lipophilicity determination of some angiotensin converting enzyme (ACE) inhibitors. Papers in another special issue on TLC of the same journal [2009, 32 (9)], also guest edited by Sherma and Fried, had papers on TLC-bioautogram analysis of vitamin B12 compounds from Japanese anchovy products; determination of the fatty acid Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
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content in butter by silver ion TLC; TLC-densitometry determination of oxyphenonium bromide and its degradation products in tablets; quantification of amino acids in the urine of BALB/c mice infected with E. caproni; analysis of selected biologically active compounds in common sage; TLC-DAD densitometry after solid phase extraction (SPE) for quantification of pesticides in water; comparison of components from red and white wines for antimicrobial activity by biodetection after OPLC (optimal performance laminar chromatography or overpressured layer chromatography) separation; validated HPTLC method for determining illegal dyes in spices; quantification of phospholipids in the lizard U. stansburiana by HPTLC-densitometry; TLC study of the stability of methyl nicotinate; reversed phase (RP) TLC behavior of some acylanilide fungicides; influence of perchlorate ion on the retention of fluoroquinolones in RP-TLC; comparison of lipophilicity determinations of organic compounds by TLC and calculations; and study of in vitro chiral conversion, phase separation, and wave propagation in aged Profen solutions. A special section of the Journal of AOAC International [2008, 91 (5)], guest edited by Sherma, featured papers on HPTLCdensitometry and HPTLC coupled with mass spectrometry (MS) and infrared (IR) spectrometry. Determinations reported were R-mangostin in Garcinia mangostana fruit rind extracts; berberine in ayurvedic formulations containing Berberis aristata; withanolides in Withania somnifera; standardization of the ayurvedic formulation Harida Khanda; three phenolic acids in Syzygium aromaticum (L.) Merr. & Perry, hederagenin in fruit pericarp of Sapindus spp.; harmine, harmiline, vasicine, and vasicinone in Peganum harmala; N-(hydroxymethyl)nicotinamide in tablets; ketorolac tromethamine in human plasma; Spirulina maxima dyes in a pharmaceutical formulation; pesticides in lake and canal water; organophosphorus pesticides in tea; neutral glycosphingolipids (GSLs) in mouse tissues; lipids in brain tissues; and lubricant additives in mineral oil. In a special section on new and improved liquid chromatography methods for food analyses of the same journal [2009, 92 (3)], edited by Morlock and Sherma, papers were included on determination of dialkyl phosphates as breakdown products of organophosphorus pesticides by HPTLC-FLD; available lysine as a measure of protein quality of fish feed by HPTLC-UV (ultraviolet) densitometry; acrylamide in coffee by HPTLC-FLD after derivatization with dansulfinic acid; and 25 water-soluble dyes in foods by HPTLC-UV-visible (UV-vis) densitometry. A special section on biomonitoring in the Journal of Planar ChromatographysModern TLC [2008, 21 (6)] contained a review article and five research papers on the use of biological toxicity or enzymatic inhibition tests combined with TLC for analysis of environmental and food samples. An article titled “Reviving Thin Layer Chromatography” (1) discussed the use of TLC to aid in sample preparation, choosing an HPLC mobile phase, and selecting regeneration or cleaning solvents for an HPLC column. A cover story titled “Moderinizing TLC” (2) reviewed the new instrumentation, materials, and analysis techniques that have made TLC a high-performance analytical method. Included was mention of the use by Bakry and Svec of photopolymerization to generate new stationary phases of 20-200 µm thickness with well-defined porous structure for 4896
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separations of biomolecules and of the development of nanoscale TLC by Morlock and Brett using plates in sizes from 1 cm × 1 cm to 4 cm × 4 cm with stationary phases from 100 to 1000 nm thick. A bibliography service (CBS) is offered by Camag Scientific Inc., Wilmington, NC, to keep subscribers informed about publications involving TLC. This service is available from Camag free of charge in paper format, or abstracts can be downloaded from their Web site (http://www.camag.ch) and searched by key word (author name, analyte, sample, technique, reagent, etc.). In addition to a review of the literature and descriptions of new products, each issue of the Camag CBS contains a section giving details of HPTLC applications, e.g., quantitative HPTLC of artemisinin in dried leaves of Artemisia annua, sildenafil determination in pharmaceutical products and aphrodisiac preparations, screening mixtures by the Bioluminex technique, and determination of acrylamide in drinking water [issue 99; September, 2007]; bioactivity based analysis of irradiated sunscreens using HPTLC and in situ detection with Vibrio fischeri, product control: bromination and oxidation of the alkaloid deoxypeganine, controlling the drying step with the ADC 2 developing chamber, quantification of β-ecdydone in a Brazilian ginseng juice, and automated HPTLC/ electrospray ionization (ESI)-MS coupling [issue 100; March, 2008]; identification and quantification of amino acids in peptides, use of RP modified precoated plates, fast identification of unknown impurities by HPTLC/MS, use of HPTLC as a problem solving technique in pharmaceutical analysis, and validation of HPTLC methods for the identification of botanicals [issue 101; September, 2008]; and a new commercial TLC/MS interface, screening for bioactive natural products in sponges, HPTLC determination of ginkogolides and bilobalide in Ginkgo biloba, and HPTLC identification of Hoodia gordonii in botanical slimming products [issue 102; March, 2009]. Many additional applications are available on the Camag Web site. Camag also publishes articles titled “CAMAGflash” on its Web site, and subscribers can automatically receive them by e-mail; those published in the review period described the progress in TLC from the early 1960s to present [February, 2008 issue]; the TLC Visualizer evaluation, visualization, and archiving system [April, 2008 issue]; training in modern TLC offered by Camag and USP (U.S. Pharmacopeial Convention Inc.) [July, 2008 issue]; method development in HPTLC [October, 2008 issue]; and the Camag TLC/MS interface [March, 2009 and August, 2009 issues]. Issue 5 of volume 21 (2008) of the Journal of Planar ChromatographysModern TLC was dedicated to the eminent Polish chromatographer Professor Edward Soczewinski on the occasion of his 80th birthday. A summary of his great accomplishments in the field of TLC was published in the issue (3). HISTORY, STUDENT EXPERIMENTS, BOOKS, AND REVIEWS A chronological history of TLC focused on the most important advances in techniques, instruments, and literature sources starting with the introduction by Runge in 1855 of paper chromatography, which strongly influenced the beginning of TLC (4), was presented by Sherma and Morlock, with input from 14 leading European and American experts in the field. The eventual
discovery of TLC by Izmailov and Schraiber in 1938 and their contributions to its development were discussed (5). TLC was used to evaluate the mechanism of dihydroxylation in a microscale student experiment for organic chemistry laboratory courses (6). Other student experiments involved the use of TLC to analyze peppermint and spearmint leaf extracts (7) and to test for unknown laboratory chemicals imitating drugs in a simulated forensic chemistry analysis (8). A book titled Thin Layer Chromatography in Phytochemistry, edited by Waksmundzka-Hajnos, Sherma, and Kowalska and published by CRC Press/Taylor & Francis in 2008, covered all aspects of the TLC/HPTLC determination of primary and secondary metabolites in plants and plant products in detail in 29 chapters and 874 pages; a companion volume titled HPLC in Phytochemical Analysis, edited by Waksmundzka-Hajnos and Sherma, will be published in 2010. The unique attributes of HPTLC compared to HPLC were highlighted in a review article that included description of two advanced techniques, bioactivity based detection and coupling with MS (9). Developments in open system planar electrochromatography (PEC) and closed system or high-pressure PEC (PPEC) were reviewed, with focus on chamber construction, equilibration of the PPEC process, separation performance in terms of time and selectivity, and general advantages, disadvantages, and prospects (10). Other review articles covered matrixassisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS directly coupled with TLC (11); TLC hyphenated with MS, Raman spectrometry, fluorometry, and IR spectrometry (12); and two-dimensional (2D) liquid chromatography including systems in which TLC is one dimension (13). Applications of TLC/HPTLC were reviewed in the following articles: polymers analyzed by thin layer size exclusion chromatography (14); resolution of polysaccharide enantiomers on chiral layers (15); plant analysis (analysis of synthetic pharmaceuticals and phytochemical (herbal) medicines and dietary supplements is the most frequently reported application) (16); aminoglycoside and macrolide residues in food matrixes (17); insecticides, herbicides, and fungicides in food, crops, biological, and environmental samples and formulations (18); identification of counterfeit drugs (19); mycotoxins in crops and foods (20, 21); acidic carbohydrates in natural products (22); ecdysteroids in plants (23); lipids (24); and foods (25). Additional published review articles are cited in the pertinent sections below. THEORY AND FUNDAMENTAL STUDIES This section contains a selection of papers reporting studies of TLC retention and lipophilicity (hydrophobicity) characteristics for a variety of compounds that were chosen as examples of very active research areas. These references also illustrate many of the different types of layers and mobile phases used for TLC and HPTLC at this time. Most of the layers used contained a fluorescent indicator that facilitated detection of compounds as fluorescence quenched zones under 254 nm UV light (F layers). The following studies of retention mechanisms and compound separations were reported: quantitative structure-chromatographic retention relationship (QSRR) studies of 30 mixed tris-β-diketonato complexes of Co(III), Cr(III), and Ru(III) on polyacrylonitrile sorbent (26); QSRR and three-dimensional (3D) molecular modeling studies of the unusual RPTLC behavior of triphenylamine derivatives on paraffin coated silica gel with 40-70% acetone-
water mobile phases (27); identification of similar and orthogonal TLC systems for 2D separations of 20 flavonoids on C-18W (fully water wettable octadecyl bonded silica gel), silica, amino (NH2), and diol modified plates with aqueous and nonaqueous binary mobile phases using the chemometric techniques principal components analysis (PCA) and heirarchical clustering (28); RP retention behavior of five angiotensin-II receptor antagonists (candesartan, eprosartan, losartan, telmisartin, and valsartin) using C-8F (octyl bonded silica gel) and C-18F layers developed in a horizontal Teflon DSII chamber and video and slit scanning densitometry for location of zones (29); effects of mobile phase buffer constituents and pH on the retention indices of 26 structurally diverse basic and neutral drugs on C-18 layers (30); QSRR approach based on functional group counts for prediction of the retention of any molecule solute in seven TLC screening systems (model optimization was performed by uninformative variable elimination elimination-partial least-squares) (31); QRRS study of 28 short peptides using linear regression analysis (32); application of electrotopological states in QSRR and quantitative structure-activity relationship (QSAR) analysis of some isomeric methyl phenols separated on C-2 (dimethyl) plates with MeOH-, ethanol (EtOH)-, n-propanol (PrOH)-, acetonitrile (ACN)-, and acetone-water binary mobile phases (33); comparison of visual and densitometric methods for characterizing the separation of phloroglucinol, niclosamide, salicylanilide, and thymol zones on silica gel with n-hexane-acetone in different volume proportions as mobile phases (the densitometric method gave smaller resolution values that were more objective and correct) (34); application of densitometry for evaluation of the separation of nicotinic acid and its derivatives using a C-18WF layer and dioxane-water (2:8) mobile phase (35); hydrophobic interaction TLC of 12 water-soluble Co(II) complexes on silica gel, cyano (CN) bonded silica gel, cellulose, and alumina layers with water-MeOH or acetone mobile phases (36); and modeling and prediction of partition coefficients of bile acids and their derivatives by multivariate regression methods on C-18W layers with MeOHwater mobile phases (37). QSAR studies using RP TLC or RP HPTLC have been often carried out to determine compounds’ physicochemical and biological properties that are related to lipophilicity for use in applications such as drug design or environmental risk assessment. Different approaches for simulating classic octanol-water shake-flask results in the measurement of lipophilicity, including RP TLC and HPTLC, were reviewed (38). In a typical TLC lipophilicity study (39), N,N-disubstituted 2-phenylacetamide derivatives were developed on C-18 layers with acetone, ACN, tetrahydrofuran (THF), dioxane, MeOH, EtOH, and 1- and 2-PrOH as organic modifiers of aqueous mobile phases; multiple linear regression analysis was used to correlate chromatographic retention constants of the compounds with physicochemical properties, and chromatographic lipophilicity (RM0; retention constant in pure water) determined by extrapolation was correlated with log P (partition coefficient) values by use of different theoretical models. Lipophilicity of parabens was investigated using C-18, C-18W, C-2, CN, and diol chemically bonded plates or oil impregnated silica gel plates with mobile phases composed of MeOH-water in different proportions and PCA of the data (40). TLC on C-18, Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
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C-18W, and C-2 plates with MeOH-, acetone-, and dioxanewater mobile phases showed that the obtained lipophilicity parameters of tauro bile acids corresponded well with theoretical values (log P virtual) (41). Lipophilicity of phenolic drugs was investigated on C-8 and C-18 layers with MeOH-water mobile phases; experimental RM0 values were compared with measured log P values and log P values calculated with seven different software packages (42). A new method of determination of log P that makes use of Rf, topological indices, and log P according to Rekker was reported for selected tocopherols; TLC was performed on C-18 HPTLC plates with EtOH and EtOH-water (95:5) mobile phases and detection of zones with methanolic 0.5% dipyridyl and 0.2% ferric chloride (1:1) detection reagent (43). CHROMATOGRAPHIC SYSTEMS Stationary Phases. TLC offers a greater variety of stationary phases than any other kind of chromatography to provide the required selectivity for a particular separation, including inorganic, organic, adsorption, partition, ion exchange, chiral, mechanically impregnated, polar and nonpolar chemically bonded phase, buffered, mixed, and gradient layers. Commercial, precoated normal phase (NP) TLC and HPTLC plates containing silica gel, an organic binder, and a fluorescent indicator on aluminum sheet, plastic sheet, or glass backing are most often used, e.g., silica gel 60F plates containing silica gel with a 60 Å (6 nm) pore diameter and phosphor fluorescing at 254 nm (designated a F254 layer by some manufacturers; the presence of the phosphor will not always be specified throughout this review when the layer is given, but it is usually present in plates with all types of layers because it allows detection of many compounds by fluorescence quenching and does not interfere with separations or other detection procedures). Reports of the use of bonded C-18 (also designated RP-18 by some manufacturers) and diol, CN, and NH2 (NP or RP depending on the mobile phase and analyte) layers are steadily increasing. This section will selectively review papers reporting analyses on layers other than silica gel. Additional diverse stationary phase/mobile phase systems are described in other sections of this review, especially the preceding one on Theory and Fundamental Studies. Multiple plates with different separation mechanisms are often required when large mixtures of compounds in complex samples must be identified and quantified; for example, HPTLC silica gel and cellulose and TLC ion exchange layers were used to determine 21 essential and nonessential amino acids in snail tissue samples (44). Gypsum layers were prepared and used to separate a variety of organic compounds, and a linear relationship between Rf values and dipole moments was found (45). Study of the retention behavior of 36 synthetic dyes on zeolite layers with n-hexane, THF, and water as mobile phases suggested that the separation capacity differs markedly from plain silica and silica coated with hydrophobic ligands (46). Layers prepared from natural diatomaceous earth that was modified by flux calcination and mixed with silica gel 60 (60 Å pore size) (1:3, 1:1,w/ w) were shown to separate Cu(II) and Co(II) complexed to sodium diethyldithiocarbamate and ammonium pyrrolidinedithiocarbamate when developed with the mobile phases toluenecyclohexane (1:3, 1:1, v/v) (47). (+)-Catechin and (-)epicatechin were determined by separation on cellulose plates 4898
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developed with water, detection with (dimethylamino)-cinnamaldehyde reagent, and densitometry at 655 nm (48). Chitosan-silica nanocomposite sorbent was described for the TLC of cytisine alkaloid and some of its derivatives with chloroform-MeOH (6:1) mobile phase, and its mechanism was considered (49). Kieselguhr developed with aqueous 0.1 M ammonium sulfate was superior to other tested systems for the selective separation of dodecyltrimethylammonium bromide from other cationic and nonionic surfactants (50). Desorption electrospray ionization-MS (DESI-MS) of peptides from tryptic digests of cytochrome c and myoglobin was carried out successfully on new 10 cm × 10 cm Merck ProteoChrom HPTLC silica gel and cellulose sheets with extra-thin 100 µm layers designed especially for one-dimensional (1D) and 2D separations of peptides (51). Catechins and theaflavins were analyzed by separation on polymaide layers using 2-fold development with chloroform-MeOH (2:3) in a horizontal chamber and detection with iron(III) chloride-EtOH reagent (52). Bonded silica layers were used in the following analyses: color pigments in wine on C-18F with ACN-water-HCOOH (40:58:2) mobile phase (53); agrimoniin and related polyphenols in pharmaceutical products on C-18W and NH2 with diisopropyl ether-acetone-HCOOH-water (4:3:2:1), THF-ACN-water (3:1:6), and acetone-HCOOH (6:4) mobile phases (54); alkaloids on CN connected to C-18 and silica plates (55); and vitamin K1 composition on C-8 and C-18 plates with methanol mobile phase (56). Ultrathin TLC (UTLC) with a 10 µm thick monolithic silica layer was shown to give faster separations and lower detection limits and required reduced sample and solvent volumes. The plates had controllable nanostructure and thickness, and the layer separation characteristics depended on the film nanostructure. Layers made with in-plane anisotropic nanostructures exhibited a decoupling effect that caused the analyte zones to not develop in the same direction as the mobile phase movement. This added layer morphology and material selection adds a degree of freedom to UTLC and may have applications in mulitdimensional TLC (57). Natural DNA was introduced as a TLC sorbent with the aim to separate chemicals like DNA binding compounds and enantiomers. By cross-linking polyvinyl alcohol with gluteraldehyde and subsequently cross-linking DNA with UV irradiation, an interpenetrating network (IPN) was formed and used to coat the surface of porous silica layer particles. High separation efficiency was found for three typical DNA binding compounds and eight amino acid enantiomers used as model compounds (58). A new “smart TLC plate” integrating a linear array of hydrogenated amorphous silicon photosensors deposited on the back of the plate was described in my 2008 review. The separation of components in a mixture can be monitored in real time by measuring the fluorescence emitted or attenuated (for nonfluorescent compounds) during the mobile phase development when the plate is irradiated with UV light. A report by the inventors was published showing the potential for quantitative analysis of mixtures of fluorescent compounds based on a linear relationship between intensity of sensor photocurrent and amount applied to the plate (59). As stated in my last review, the wide ranging practical value of these plates remains to be demonstrated.
Silver impregnated silica gel layers (argentation TLC) continue to be widely used for separations of fatty acids. The following representative analyses were reported: conjugated linolenic acids in bovine milk and muscle (60), modified fatty acids and sterols in processed fats and oils (61), and trans-octadecenoic acid and conjugated linoleic acid isomers in Canadian dairy products (62). Nicotinic acid and its isomers were separated on silica gel 60 and kieselguhr plates impregnated with 2 and 5% aqueous copper sulfate with n-hexane-acetone mobile phases (63). RP TLC separations of nonionic and cationic surfactants were performed on silica gel layers impregnated with 5% paraffin oil, silicone oil, and tributyl phosphate using water and polar organic solvents as mobile phases (64). Nonprotein R-amino acids were derivatized with Marfey’s reagent and its four variants, and separation of the resulting diastereomers was carried out by RP TLC on C-18WF plates with mobile phases composed of ACN-MeOH + triethylammonium phosphate buffer (65). To avoid derivatization, chiral analyses are often carried out on silica gel layers impregnated with chiral selectors. For example, (±)-ketamine and (±)-lisinopril by use of (+)-tartaric acid or (-)mandelic acid as impregnating reagents or mobile phase additives (66); atenolol and propranol enantiomers using L-tartaric acid, (R)mandelic acid, and (-)-erythromycin as chiral selectors (67); and enantiomers of six β blockers with L-aspartic acid and L-glutamic acid as impregnants and ACN-MeOH-water-dichloromethane (DCM) and ACN-MeOH-water-glacial acetic acid (HAc) mobile phases in different proportions (68). Mobile Phases. Mobile phases are usually chosen after guided trial and error testing of reported solvent mixtures with appropriate strengths (Rf values of 0.2-0.8 are optimum) and selectivities relative to the layer and mixture to be separated. In addition, various systematic, computer based optimization procedures have been proposed, such as PRISMA and Snyder’s solvent classification. The role of mobile phases in controlling selectivity for adsorption thin layer or column chromatography was reviewed by Snyder. For a binary mobile phase (A/B), the choice of the more polar component (B) largely determines relative retention and resolution. Maximum differences in selectivity are achieved when B is either very polar (low %B needed) or relatively nonpolar (high %B needed). Further (smaller) changes in relative retention can be achieved by the use of mobile phases for which the B solvent differs in hydrogen bond basicity. A seven mobile phase experimental design is offered for optimization of the mobile phase composition (69). Cyclodextrin derivatives were used as mobile phase chiral selectors for analysis of the (R) and (S) enantiomers of the anesthetic bupivacaine on HPTLC silica gel plates (70). Binary homogeneous azeotropic mixtures of solvents with different polarity were successfully used to separate various fluorescent dyes (71). A total of 28 different salts were studied as mobile phases for salting out TLC on silica gel of Co(III) complexes (72). Aqueous methionine mobile phase provided identification of the cationic surfactant cetylpyridinium chloride (CPC) on a kieselguhr layer with preliminary separation from nonionic surfactants (73). CPC containing mobile phase provided separation of Zn(II), Cd(II), and Hg(II) cations on silica gel (74), and N-cetylN,N,N-trimethylammonium bromide (CTAB) containing phases
were used for identification of diclofenac sodium from human urine on silica gel H (containing a small amount of colloidal silica gel but no other binder) (75) and the RP separation of paracetamol, ibuprofen, diclofenac, aspirin, and ascorbic acid on various impregnated silica gel H layers (76). A large number of amino acids were separated and analyzed using the following new mobile phases: organic-aqueous systems modified with 10-80 mM of neutral and chaotropic salts (chlorides, iodides, nitrates, thiocyanates, perchlorates, and hexafluorophosphates) on cellulose (77); buffered aqueous mobile phases on silica gel 60 for 27 amino acids (78); CTAB-n-butanol (BuOH)-n-octane-water microemulsion on silica gel (79); 1.0 × 10-5 M Triton X-100-8.1 × 10-4 M sodium dodecyl sulfate-acetone (1:1:5) on silica gel (80); and 1-BuOH-aprotic organic solvent-micelles mixtures on silica gel (81). Additional stationary and mobile phase combinations are specified for separations reported in other sections of this review. APPARATUS AND TECHNIQUES Sample Preparation. In TLC, the complete sample is contained in the chromatogram between the origin and the mobile phase front at the top of the plate. Because the plate is not reused, strongly retained components at the bottom are no problem, whereas in an HPLC run the strongly retained sample components may be irreversibly sorbed on the column and change its performance for later runs or they may elute later and interfere with subsequently injected samples. Therefore, sample purification (cleanup) is more critical in HPLC compared to TLC. The usual sample preparation for TLC still involves simple dissolving; traditional extraction in a flask, separatory funnel, or Soxhlet apparatus or by refluxing; and cleanup by liquid-liquid partitioning if required. Some liquid samples, such as beverages or oils, can be applied to the layer directly with no sample preparation or after simple dilution with a solvent. Additional sample preparation methods are mentioned in many of the applications described below. Protein precipitation with ACN is a common method for cleanup of biological samples, and it was used in the determination of the analgesic aceclofenac in plasma by densitometric scanning at 270 nm after separation on silica gel 60F with toluene-ethyl acetate (EtAc)-MeOH-HAc (7:2:2:0.1) mobile phase (82). Carbon sorbents were applied for extraction of impurities from 3,4methylenedioxymethamphetamine (MDMA, the main psychotropic component of “ecstasy” tablets) prior to analysis by silica gel TLC with chloroform-MeOH-ACN (5:2:3) mobile phase and UV detection (83). Supercritical fluid extraction (SFE) with carbon dioxide was used for the extraction of active sex hormones, including estradiol and progesterone, from antler velvet (84); lipids from cheese (85); ceramides from wool (86); and flavonoids from black, green, and red teas (87) prior to TLC analysis. Biologically active compounds in rhizomes of Rhodiold rosea L (88) and acrylamide in drinking water (89) were quantified by TLC-densitometry after SPE. Thin Layer Chromatography. Instruments for modern TLC were described in a Field Guide to Instrumentation article (90). The vast majority of TLC analyses are carried out by a single capillary flow ascending development in a mobile phase vapor saturated large volume chamber (N-chamber) or Camag twin trough chamber (44) at ambient laboratory temperature. The latter Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
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is an N-chamber modified with an inverted V-shaped ridge in the bottom dividing the tank into two sections that allow development with less mobile phase as well as equilibration with the vapors of different solutions if desirable. Automatic ascending development in the Camag ADC2 with controlled conditions of mobile phase distance, chamber saturation and humidity, and final drying is used when methods must comply with good manufacturing practice (GMP)/good laboratory practice (GLP), installation qualification (IQ)/operation qualification (OQ), and 21 Code of Federal Regulations (CFR) Part 11 requirements. In addition, horizontal development in the Chromdes Teflon DSII chamber is quite often reported in the literature (e.g., ref 29). Methods of obtaining optimum separations for circular and linear TLC by applying focusing liquids to the initial zone were described (91). Application of a microwave field arranged perpendicular to the TLC plate resulted in improved separations (92). A simple temperature controlled horizontal chamber for nonforced flow microplanar NP and RP TLC was described for isothermal or gradient developments from -20 to 80 °C in 5-20 min using 0.3-1.0 mL of mobile phase and applied to separations of cyclodextrins, fullerines, and steroids (93). 1D multiple development with the same or different mobile phase is a method for improving resolution in capillary flow TLC. Photodegradation products of atorvastatin were separated by double development on silica gel with EtAc-n-hexaneHAc-MeOH (40:55:0.5:4.5) followed by 39:55:0.5:5.5 in a stability indicating assay (94). Automated multiple development (AMD) is carried out in a Camag chamber with increasing development lengths and an isocratic mobile phase or a mobile phase gradient with decreasing strength; bioactivity based analysis of sunscreens was carried out using automated spray-on band application with a Camag Linomat 5, AMD (seven step gradient of tert-butyl methyl ether-n-hexane), and detection by UV light in addition to V. fischeri in a Camag Bioluminizer (95). A larger zone capacity for complex mixtures can also be obtained by 2D (or bidimensional) TLC. In the usual 2D method, a single sample is spotted at the corner of a layer that is developed in orthogonal directions with two mobile phases having complementary separation mechanisms (with drying between developments) or by the use of two layers with complementary mechanisms. A disadvantage of 2D TLC is that quantitative densitometry is not possible by the preferred method of comparing standards and samples developed identically on a single plate; standards must be developed in two dimensions on one plate and compared to samples on a different plate, or standards are applied to the edge of a plate before each 1D development, in which case the standards applied after the first development are not developed in both dimensions as are the analytes to which they are compared. An example of the use of a single layer was the simultaneous separation of curcumin, metanil yellow, Sudan I, and Sudan IV in tumeric, chili, and curry powders by development of a silica gel layer with chloroform-methanol (9:1) followed by toluene-hexane-HAc (50:50:1) (96). Graft TLC with different layers was used separation of coumarin fractions present in fruit extracts; the method involved triple development of mixtures on a CN layer with ACN-water (30:70) (RP TLC), cutting the layer into strips containing partly separated mixtures in the direction of development, connection of each strip to a silica gel layer by 4900
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clamping, transfer of zones to the silica gel by elution with MeOH, and triple development of the silica gel layer with EtAc-n-hexane (35:65) (NP TLC) (97). A new 2D technique with multiple gradient development (MGD) instead of isocratic development in each dimension was carried out using a single 10 cm × 20 cm silica gel plate in a horizontal DS chamber for analysis of plant extracts; two samples were applied near the side edges and developed in the 10 cm direction with a three step gradient of hexane-DCMEtAc-MeOH-HCOOH (80%), then the plate was developed with a two step gradient of the same solvents without MeOH from the side edges toward the center in the 20 cm dimension (98). Use of equal spreading criteria was proposed for selection of the two most orthogonal (complementary in terms of selectivity) mobile phases for 2D TLC (99). Techniques of 2D TLC were reviewed for analysis of secondary plant metabolites, including use of different mobile phases with one layer, bilayer plates, graft TLC, and combinations of different multidimensional techniques (100). OPLC is a forced flow method performed in an instrument (OPLC-NIT Ltd.) in which the plate containing an optimized HP layer is covered with a flexible, inert polymer sheet under pressure and mobile phase is pumped through the sorbent layer. In effect, the layer is analogous to a flat HPLC column. The OPLC 50 system with the four channel flowing eluent wall (FEW)/inlet/outlet (I/ O) configuration was shown to be suitable for fully offline parallel separations and for four channel fully online separations; fully online separations can be performed with aliquot detection, a new type of online detection of partially mixed samples in which the regions where mixing of parallel samples occurs are excluded (101). Published determinations by OPLC included xanthine compounds with online DAD and MS hyphenation (102); basic drugs in autopsy liver samples by dual plate OPLC (103); eight carbohydrates in fermentation medium by over-running elution with acetone-ACN (85:15) on HPTLC silica gel (104); volatile organic compounds (VOCs) recovered from peppermint by solvent extraction and SFE (105); and flavonoids recovered by open vessel microwave extraction (OVME), decoctum, infusion, and ultrasound extraction from plant material (106). PPEC is a planar chromatographic method in which the mobile phase is driven by electroosmotic flow while the system is pressurized. Characteristics of PPEC were described in terms of instrument design and variables influencing separation efficiency such as mobile phase composition and pH, buffer concentration, applied voltage, plate preparation and type, and temperature (107). PPEC was shown to be applicable for determination of solvent composition-retention relationships in RP systems (RP-18W HPTLC plate) (108). PPEC with Macherey-Nagel Chiralplates having D-4-hydroxyproline as the chiral selector was used with mobile phases prepared from ACN, MeOH, water, and buffer solutions for separations of the enantiomers of tryptophan and valine that were superior to those obtained by TLC (109). Three modes of sample application were compared for PPEC, and prewetting of the plate followed by application with a commercially available aerosol applicator gave the best separation performance (110). Nine analytes were separated by PPEC and detected by direct analysis of the RP-18WF plate using DESI coupled to MS/ MS with a total analysis time of 10 min and minimal sample preparation (111); the instrument used to perform PPEC and control of experimental variables such as pressure, temperature,
and soaking of the plate in the mobile phase to improve reproducibility were described in an earlier paper (112). Planar dielectrochromatography (PDEC) is another method that uses an electric current like PPEC, but the separation selectivity is improved by using suitable transverse alternating electric fields to modify the mobile phase front velocity and migration distance of solutes with the plate in a vertical chamber (113). TLC has been used over the years to aid in selecting mobile phases and predicting separations for column chromatography. Peptide retention in RP HPLC was predicted with the use of amino acid retention data obtained in a QSRR study on silica gel plates developed with water-ethanol (2:8) (114). Given sample mass and TLC data, a spreadsheet was developed that provides information on the amount of silica gel needed, the optimal fraction size, and degree of separation to be expected for preparative scale flash chromatography separations (115). A simple protocol was described for estimating SPE elution volumes of steroids based on retention data generated from microplanar chromatography with C-18W plates and mobile phases composed of methanol-water mixtures ranging from 20 to 100% (116). DETECTION AND IDENTIFICATION OF SEPARATED ZONES Zone detection in TLC is based on natural color, fluorescence, or UV absorption (fluorescence quenching on phosphor-impregnated F layers) or on the use of various universal or selective chemical or biological detection reagents applied by spraying or dipping. Densitometric scanning in fluorescence and absorbance modes is an important detection and documentation technique, as well as for quantitative analysis. A great advantage of TLC lies in the ability to use a number of detection methods and reagents in sequence on a single layer storing the entire sample in the chromatogram to increase the amount of information obtained; analytes do not necessarily have to be separated in order to obtain their successive quantification by use of different compatible detection methods. Zone identification is initially based on the correspondence of Rf values and detection characteristics between sample and standard zones but must be confirmed by other evidence, such as offline or online coupling of TLC with various spectrometric methods. The ability to apply multiple detection methods without the time constraints of column elution chromatography, the need for less sample cleanup, and the high throughput and resultant speed and economy offered by the simultaneous analysis of many samples on a single layer with appropriate standards are important advantages of TLC and HPTLC. Additional detection and identification methods are cited in other sections of this review. Chemical Detection. A simple, inexpensive, compressed air operated detection reagent spraying device that can handle microliter to milliliter volumes of corrosive liquids with a wide range of viscosity was constructed (117). Spraying, dipping, and use of the AR2i Derivapress device were compared for application of phosphomolybdic acid (PMA) reagent in the detection and quantification of neutral lipids by silica gel HPTLC (118). A study of the UV imaging of zones with a charge coupled device (CCD) camera on TLC aluminum backed plates while still wet indicated that monitoring of separations by UV absorbance in real time was feasible (119). Validation of results is usually carried out for quantitative densitometric determinations, but procedures were
described for specificity, reproducibility, and robustness validation of the qualitative identification of Hoodfia gordonii according to cGMP (current GMP) requirements (120). The following are reported studies of new and established detection reagents. Anisaldehyde and crotonaldehyde detected sesame oil unsaponifiables as brightly colored zones (121). Steroids separated by silica gel OPLC were detected using sulfuric acid, PMA, and phosphoric acid reagents (122). Glucosamine in nutritional supplements was densitometrically quantified using detection by simple heating of an NH2 plate after development to produce a derivative that can be detected either by absorption (25 ng sensitivity) or fluorescence (15 ng) (123). Benzil and benzoin were found to be general spray reagents for visualization of organic compounds and natural products as zones with various colors characteristic of different classes (124). Diazepam was specifically detected among other benzodiazepine drugs as violet zones with a sensitivity of 5 µg in biological and nonbiological samples using NaOH-m-dinitrobenzene reagent (125). 3,4-Methylenedioxy-methamphetamine and five related compounds were detected in urine samples with a detection limit of 50 ng as blue fluorescent zones by spraying with a reagent consisting of sodium hypochlorite, potassium hexacyanoferrate(III), and NaOH and heating at 110 °C for 3 min (126). Dipeptides were derivatized with phenyl thiocarbamyl to form sulfur containing compounds that could be detected as white zones on a violet-gray background at picomole levels by spraying the silica gel plate with sodium azide and starch and exposure to iodine vapor (127). Stearic acid, stearyl alcohol, and methyl stearate were detected more successfully on silica gel 60 with the dyes gentian violet, methylene violet, methylene blue, methyl green, malachite green, and Janus blue compared to the usually applied Rhodamine B (128). 2,4,6-Triphenylpyrylium salts were transformed into the yellow or blue parent thiopyrylium derivatives upon reaction with zones containing sulfide ions at the origin before mobile phase development; the limit of detection (LOD) for sulfide was picomole/zone (129). The best detection of ergosterol, stigmasterol, and selected steroids on silica gel by PMA reagent was accomplished by heating at 40-80 °C for more than 20 min instead of the more common shorter heating at higher temperature (130). Sucralose in dietary products was quantified at low milligrams/100 g concentrations by densitometry on HPTLC silica gel after postchromatographic derivatization with 2-naphthol sulfuric acid reagent (produced a brown zone scanned at 500 nm) or aniline diphenylamine orthophosphoric acid reagent (gray blue zone scanned at 366/>400 nm) (131). Biological Detection. The principle of bioautography is that a suspension of a microorganism growing in a suitable medium is applied to a developed TLC plate after drying; incubation of the plate with the microbes in a humid atmosphere at the optimum temperature allows growth of the bacteria; and, by use of a specific dye, live cells can be visualized because, e.g., dehydrogenases from living microorganisms convert a tetrazolium salt into intensely colored formazan. The antibacterial compounds appear as colorless zones against a colored background. The BioArena system integrating TLC or OPLC with bioautography was used for indirect detection of endogenous ozone in zones (132) and to Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
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study the mechanism of action of paclitaxel (133) and the influence of Pseudomonas bacteria on aflatoxin B1 (134). TLCbioautography was also applied to the molecular screening of acetylcholinesterase inhibitors of natural origin (135), study of the antimicrobial activity of two species of the genus Trametes Fr (136) characterization of vitamin B12 compounds from Korean purple laver (Porphyra sp.) products (137), and evaluation of the antibacterial activity of essential oils of Zataria multiflora, Carum copticum, and Thymus vulgaris (138). Bioluminescence detection is carried out in the Bioluminizer using V. fischeri to coat the plate, incubation under controlled conditions and detection of toxic substances as dark zones on a luminescent background under UV light. The principles, instrumentation, techniques, and applications of bioluminescence detection in TLC were reviewed (139). TLC on silica gel 60F HPTLC plates developed with toluene-ethyl formate-HCOOH (5:3:2) was used with the Bioluminizer to detect adulteration of the phytomedicine Actaea racemosa (140). TLC with immunodetection (immunostaining) was used to quantify plasma fatty acids in a study of the prevention of osteoporosis in Cftr-KO mice by fenretinide (141), analyze GSL expression in cerebrospinal fluid of infants with neurological abnormalities (142), show that an antibody secreted by a hybridoma binded to sulfated glycosides but not to neutral sphingolipids (143), study Lewis antigens and H-pylori adhesion in CHO cell lines engineered to express Lewis b determinants (isolated glycolipids were analyzed) (144), and identify the GSLs accumulating in the nervous tissues of mutant mice upon in 9-Oacetyl GD3 expression (2D TLC) (145). Thin Layer Chromatography/Mass Spectrometry. A continuing very high level of research in the techniques and applications of TLC/HPTLC coupled with MS is one of the major trends in the past 2 years, and it is anticipated that these research efforts will increase in the future, especially now an interface is commercially available from Camag. Plungers with circular or oval edge geometries were compared for HPTLC/MS coupling via the ChromeXtract interface in the analysis of Glu-P-1 and harmane, and each showed different advantages; this interface is now sold as the Camag TLC/MS interface (146). High-resolution MS (HRMS) directly from TLC plates was reviewed with emphasis on application to low volatility, low thermal stability compounds, low sample preparation requirement, and real time monitoring of chemical reactions in organic synthesis (147). Easy ambient sonic spray ionization TLC/(EASI)MS/MS was shown to be advantageous for drug, oil, phytochemical, synthetic chemical, and forensic analyses (148). A comparison was made between two HR, surface based, MS methods, TOF secondary ion MS (TOF-SIMS) and MALDI TOF-MS, for determining abietic and gibberellic acid molecular profiles on different types of silica gel plates (149). The following TLC/MS analyses were reported: tumor associated GSLs in hepatocellular and pancreatic cancer by IR-MALDIorthogonal (o)-TOF MS coupled with TLC binding overlay analysis (150); biodiesel and petrodiesel-biodiesel blends by HPTLC/EASIMS (151); egg yolk lipids by HPTLC/MALDI-TOF-MS (152); pharmaceutical formulations by DESI ion mobility MS from nonbonded RP TLC plates (153); chromium species by HPTLC/ laser ablation inductively coupled plasma (ICP) MS (154); dyes, 4902
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amines, and drugs using electrospray assisted laser desorption/ ionization (ELDI) MS (155); cyanobacteria toxins by HPTLC-UV spectrometry/MALDI-o-TOF/MS (156); tryptic protein digests by 2D TLC/DESI-MS (157); phospholipid mixtures by HPTLC/ MALDI-o-TOF/MS (158); human plasma lipids and soybean lecithin by HPTLC/MALDI-MS (159); and lipids in the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana by TLC/MALDI-TOF MS (160). Thin Layer Chromatography Coupled with Other Spectrometric Methods. In addition to the interfacing with UV spectrometry listed above (156), TLC was coupled with surface enhanced Raman spectrometry (SERS) for analysis of artist dyestuffs on silica gel coupled with citrate reduced silver colloids (161) and diterpenoic acids on silver colloid treated layers with a modified aluminum backplate (162). Sample Identification Software. Ballpoint pen inks extracted from documents with MeOH were compared and identified using TLC separation on silica gel 60 plastic backed layers with EtAc-EtOH-water (73:35:30) mobile phase, scanning of chromatograms by an ordinary office scanner, and image analysis using specific software for evaluating thin layer chromatograms (TLS-IA) based on the intensity profile of red, green, and blue (RGB); a discriminating power of 92.8% was found for inks from different manufacturers based of patterns of the RGB profiles (163). Camag introduced a visual image comparison software, the Planar Chromatography Manager, that was shown to be especially applicable in quality assurance (QA) fingerprint analysis for identification of botanicals and detection of adulterants in botanical raw materials and finished products, as well as for analysis of forensic, food, and environmental samples. QUANTITATIVE ANALYSIS Modern quantification in TLC and HPTLC involves the use of a densitometer after sample preparation, automated application of standards and samples to the layer usually as a band (zones in the shape of bands can be better resolved than round spots), mobile phase development of the layer, and detection of the analyte zones. The densitometer scans a series of standard zones, a calibration graph is generated from the peak height or area vs weight by linear or polynomial regression, and the weights of bracketed sample zones are interpolated from the graph. Internal standardization is only occasionally used in HPTLC-densitometry, most commonly in clinical drug analyses. Slit scanning densitometers are most applied by far, but camera based (29), DAD, and office-type computer scanner densitometers (163) are also used. Techniques and Instruments. A Linomat 5 applicator and Camag 3 slit scanning densitometer were used in the analysis of 2-arylpropionate derivatives in pharmaceutical preparations on silica gel 60F using two mobile phases; scanning was carried out at 225 and 270 nm, and recoveries ranged from 98.7 to 102% (164). Quetiapine was determined in tablets using HPTLC on silica gel and C-8 plates and a Desaga VD-40 videodensitometer with ProQuant image capture and analysis software; no significant differences in validation results were observed when compared to a Desaga CD-60 slit scanning densitometer (165). Patulin in apple juice was quantified by EtAc extraction, sodium carbonate partitioning cleanup, and FLD with a CCD camera; recovery was 95%, LOD 5 ng/zone, and limit of quantification (LOQ) 14 g/L (166). Images acquired with a standard digital camera under 365
nm UV light and image analysis software based on a developed algorithm were shown to provide validated quantification of thin layer chromatograms, as demonstrated by the assay of cichoric acid present in Echinacea purpurea L. on a polyamide plate with chloroform-MeOH-HCOOH-water (3:6:1:1) mobile phase (167). Analysis of TLC images from a digital camera showed that the technique is low cost and efficient for qualitative and quantitative analysis, capture in color is more sensitive than black and white, 3D visualization is easy to achieve by use of OpenGL technology, and sensitivity was increased by use of longer acquisition times but this reduced the linearity of quantitative analysis (168). Validated quantitative analysis of caffeine from pharmaceutical preparations on HPTLC RP-18W plates with MeOH-HAc-water (25:4.3:70.7) mobile phase was obtained using a digital camera and Sorbfil TLC software (169). Curcuminoids in tumeric were simultaneously determined in the range of 0.375-6 µg/zone using a digital camera and public domain Scion software (170). The Tidas 2010 fiber optics DAD instrument allows in situ scanning of spectra and areas of zones in chromatogram tracks in the range of 200-1100 nm. Absorption DAD TLC was used for determination of melamine in diluted milk by application of bands with a Desaga AS 30 to aluminum backed silica gel 60 layers, development with 2-PrOH-DCM-water (3:1:1), and detection with Wuster’s blue or iodine-starch reagent (171). Quantification of dequalinium cations in pharmaceutical samples was carried out by absorption and fluorescence DAD TLC with Kubelka-Munk transformation on Merck Nano TLC silica gel plates with the mobile phase MeOH-7.8% aqueous ammonium acetate (17:3) (172). C-18 SPE and TLC on silica, C-18, CN, diol, and NH2 layers in a horizontal DS chamber with DAD densitometry were used for qualitative and quantitative analysis of dyes in beverages (173). The Ar2i ChromImage flatbed scanner with Galaxie software is a system specifically designed to capture and analyze images of chromatograms on thin layer plates. However, most reported applications of densitometry with a PC scanner involved use of a simple office scanner with separate software. The ChromImage in the visible mode was used to quantify β-carotene in a dietary supplement on a C-18 layer developed with petroleum ether (PE)-ACN-MeOH (1:1:2) (174). Food dyes were determined in different products by TLC on an NH2 bonded layer with isoPrOH-DE-ammonia (2:2:1) mobile phase and densitometry using a flatbed HP ScanJet 3970 office scanner with MachereyNagel TLC scanner software (175). An HP M1005 MFP Laser Jet office scanner with ImageQuant TL v. 2003 image analysis software was used in the silica gel TLC quantification of steroid drug intermediates formed during bioconversion of soysterols with detection by spraying with cerium ammonium sulfatesulfuric acid reagent (176). Validated TLC image analysis for simultaneous quantification of curcuminoids that were MeOH extracted from Curcuma longa was carried out on silica gel using chloroform-hexane-MeOH (1:1:0.1) mobile phase, an HP Scan Jet 3500C digital scanner, and Photoshop 7.0 software (177). Prostaglandins PGE2 and PGF2R were detected by postrun derivatization with PMA on silica and C-18 layers and robustly quantified using a Plus-tek OpticPro S12 USB office scanner with Image Folio v. 4.2.0 image acquisition software and Scion Image freeware (v. 4.0.3.2) (178).
Komsta compared different algorithms for denoising thin layer densitograms (179) and videoscans (180, 181). The importance of using a carefully analyzed standard in densitometry was shown for the analysis of lycopene by silica gel HPTLC with visible mode densitometry; use of a ChromaDex lycopene standard gave correct results while two other commercial standards were found to be very impure compared to their stated values and gave very high results when used for sample analysis (182). TLC/HPTLC quantitative methods must be validated to prove the reliability of the obtained results. Factors considered may include accuracy (recovery), precision [repeatability and intermediate precision in terms of relative standard deviation (RSD)], specificity, linearity, range, LOD, LOQ, and robustness. The International Conference on Harmonization (ICH) has published guidelines for validation of pharmaceutical analyses based on characteristics such as these, and most published TLC analyses use all or part of the ICH guidelines for quality assurance of results, especially those for determinations of drugs. As examples, ICH guidelines were applied for validation of HPTLC-densitometry methods for the immunosuppressant tacrolimus in formulations (183) and the nonsteroidal antiandrogen bicalutamide in bulk drug and liposomes (184). An interlaboratory study (14 laboratories in five countries) was performed to validate an NH2 layerdensitometry method for determination of sucralose in carbonated and still soft drinks at proposed European Union (EU) regulatory limits (185). Applications. In addition to the many quantitative analyses already cited, this section presents selected, important examples of a variety of analytes and sample types for which new or improved densitometic methods have been reported in the review period. Unless stated otherwise, it should be understood that automated instrumental sample application as bands or spots, an HPTLC silica gel F layer, a single ascending (vertical) 1D mobile phase development, fluorescence quenching detection, and measurement of peak areas with a slit scanning densitometer in the reflectance-absorption mode at the maximum absorbance wavelength between 200 and 286 nm were used in each entry. Validation data are given for many of the applications to document the excellent quantitative results that are possible using modern HPTLC, and the dominance of the number of methods published for pharmaceutical and herbal medicine and dietary supplement analysis is illustrated by the papers selected for inclusion. Miscellaneous Analytes. Neutral lipids (NLs) (186, 187) and polar lipids (PLs) (188) were determined in human urine and urine of BALB/c mice that were infected with E. caproni or uninfected. Lipids were extracted with chloroform-MeOH (2:1), mobile phases were PE-DE-HAc (80:20:1) for NLs and chloroform-MeOH-water (65:25:4) for PLs, and zones were detected with PMA (NL) or cupric sulfate (PL) reagents. Tocopherols were quantified in vegetable oils using SPE with Porapak P, chloroform mobile phase, bipyridyl-ferric chloride detection reagent (pink zones), and densitometry using a CanoScan Lide 20 flatbed scanner with 600 × 1200 dpi resolution. R-Tocopherol was found to be present in sunflower, olive, corn, soy, and almond oils at 405-680 mg/L levels; γ-tocopherol in corn and soy oils at 419 and 479 mg/L, respectively; and δ-tocopherol in none of the oils (189). Analytical Chemistry, Vol. 82, No. 12, June 15, 2010
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The antibiotics bacitracin and nystatin have been banned by the EU as additives in animal feed. A routine HPTLC quality control (QC) method in support of this regulation was developed using a C-18 F plate and a mobile phase containing ACN, toluene, MeOH, and pH 2.5 KH2PO4 buffer. Respective recoveries were 98.7 and 105%; intermediate precision was below 2.2% for both analytes; and LOD was 4.5 and 5.5 ppm, respectively (190). Glycoalkaloids (R-solanin and R-chaconine) were determined in potatoes during processing by homogenization with MeOH-HAc (95:5), horizontal development with DCM-MeOH-2.5% aqueous ammonia (70:30:4.4), detection by dipping into modified Carr-Price (antimony trichloride) reagent, and densitometry at 560 nm. LOQ was 30 ng/zone, LOD 5-20 ng/zone, and linearity range 30-700 ng (191). The same two compounds were determined in peeled potato tubers by extraction with 1% HAc in water containing 1-pentane sulfonic acid, C-18 SPE cleanup, development with the DCM-MeOH-ammonia (70:30:0.5), detection with Ce(IV) reagent, and scanning at 505 nm. Recovery for the compounds was 80-90% for 20-100 mg/kg, LOQ was 5.0 mg/kg, and respective repeatability values (RSD) were 8.2 and 11.4% (192). Four phthalate esters extracted with DCM from lake water samples were determined using optimized one to three component mobile phases, ascending and horizontal development with controlled humidity, and scanning at 202 nm. Validation values at 0.5-5 µg/L concentrations were LOD 7 ng, LOQ 0.1 µg/L, >80% recovery, interday precision
