APPLICATION SPOTLIGHT

ABSTRACTThis application note presents a simple, user-friendly technique for a comprehensive two-dimensional analysis of complex matrices when the operator only has access to a one-dimensional liquid chromatography system. In this example an 1260 Agilent Infinity chromatograph coupled to a fraction collector was used for an offline study of biochemical components of mushrooms; however, the approach is easily transferable to other sample types since all that is required is a standard Agilent one-dimensional HPLC system (consisting of an autosampler, degasser, a binary or quaternary pump, thermostatted column compartment, a diode array detector, a fraction collector) and appropriate data processing software.

INTRODUCTIONThe analysis of complex matrices such as natural/food products, petrochemicals and many environmental and biological samples is crucial in many areas of both industrial and academic research. The main aim of such investigations is usually the identification of particular compounds of interest, such a specific contaminant (or group of contaminants) that may be present in conjunction with a multitude of other substances, or simply the separation and quantitation of the components of a particular sample. These types of analyses require chromatographic techniques with high peak capacity in order to achieve the separation of as many compounds

Jessica Pandohee,1 Paul G. Stevenson,2 Xavier A. Conlan2 and Oliver A.H Jones1* 1School of Applied Sciences, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia 2School of Life and Environmental Sciences, Deakin University, Waurn Ponds, Geelong, VIC 3216, Australia* Author to whom correspondence may be addressed

E-mail: Oliver.Jones@rmit.edu.au Phone: +61 3 9925 2632 Fax: +61 3 9925 3747

Two-dimensional HPLC analysis of complex matrices using a standard 1260Agilent Infinity LC system.

as possible. This is a challenging task since the samples often consist of thousands of compounds from many differing classes, which may vary greatly in factors such as concentration, mass and polarity. The analysis of many compound classes is further complicated by the fact that many of their constituent members are isomeric. A pertinent example are the sugars, for instance glucose, galactose and mannose are isomeric aldohexoses while arabinose and xylose are aldopentoses and sorbitol is the aldehyde-reduced derivative of glucose [1]. With the exception of sorbitol, these compounds exist in solution mainly in ring-closed form with various ratios of pyranose (6-membered) to furanose (5-membered) rings. The difficulties described above often result in co-elution of closely related compounds and/or insufficient separation during chromatographic analysis (peak overlapping), resulting in poor resolution of compounds of interest and in some cases masking of compounds of low concentration if/when they co-elute.

A solution to the analysis of complex samples is the use of multidimensional liquid chromatography; this technique uses two columns with differing phases. A sequential collection of aliquots is made from the first column (the first dimension) and reinjection onto a second column (the second dimension), the resulting data are then plotted in either 2D or 3D space. The total peak capacity of such a system is the product of the combined peak capacities of each dimension. However, two-dimensional LC-LC systems are new technology and can therefore be complex and expensive to set up. The price for such equipment may be up to US$130,000 and users may need to undergo extra training and become acquainted with new software and a complicated and unfamiliar set up.

It is however possible, to perform a two dimensional analysis using only a standard one-dimensional HPLC system. This format also has some advantages in that the second dimension is independent of the first, which simplifies the optimisation of the

Sample Preparation

First Dimension

Fraction Collection

Second Dimension

Data Analysis

Crush sample (1 g) under liquid nitrogen

Boil for 1.5 h with 300 mL ethyl acetate

Remove (and keep) solvent, repeat extraction with a further 300 mLof ethyl acetate for 1 h

Combine extracts and evaporate to dryness under vacuumat room temperature

Reconstitute in 1 mL of methanol and filter through 0.45 µmsyringe filter before analysis

Run sample on CN column twice, each time injecting 100 µL andcollecting in 250 µL clear plastic vials

Set fraction collector conditions to 0.1 min/tube with start timebefore the first peak is eluted and stop time just after the last peak is

eluted (in this case start time is 0.6 min and stop time is 5.6 min)

Ensure that there are no air bubbles in vials, run all collectedfractions as a sequance on C18 column

Export data as CSV, plot using Mathematica

EXPERIMENTALSample preparationThe sample preparation procedure used in this study is outlined in figure 1. A methanol/chloroform/water extract [2] may also work for less robust sample types e.g. blood.

Figure 1. Flow chart for non-automated two dimensional liquid chromatography analysis

ChemicalsAll mobile phases were HPLC grade solvents. Milli-Q water (18.2 MΩ) was obtained in-house. Methanol (Isocratic, HPLC-grade 254 nm) was purchased from Scharlau Chemie (South Australia, Australia) and acetonitrile from Ajax Fine Chem (New South Wales, Australia). All mushrooms were purchased from a local supermarket in the Melbourne CBD.

first dimension parameters); additionally, the second separation is not time limited meaning longer columns with greater peak capacity can be utilised, resulting in a larger total peak capacity than is possible using a short columns in the second dimension; N.B. Short, second dimension columns are required in an automated 2D LC-LC system since such instruments require the separation of a sub-sample in the second dimension to be completed before the subsequent sub-sample from the first dimension is injected.

There are therefore clear advantages in both cost and resolution to using a standard HPLC to perform two-dimensional separation. This application note outlines the steps for the method development of a reversed phase, non-automated, comprehensive two-dimensional liquid chromatography analysis. The button mushroom (Agaricus bisporus) is used as a model sample in this case but the technique is easily transferred to any other sample matrix.

ColumnsFirst dimension Phenomenex Luna CN, 150 mm x 4.6 mm x 5 µm (run at room temparature)Second dimension Phenomenex Kinetex C18, 150 mm x 4.6 mm x 5 µm (run at room temparature)

First dimension pumpSolvent A WaterSolvent B MethanolFlow rate 1 mL/minGradient 5% B at 0 mins; 100% B at 3 mins; 100% B at 6 minsStop time 6 mins

Second dimension pumpSolvent A WaterSolvent B AcetonitrileFlow rate 3 mL/minGradient 5% B at 0 mins; 100% B at 1 mins; 100% B at 4 minsStop time 4 mins

AutosamplerInjection volumn for first dimension 100 µLInjection volumn for second dimension 100 µL (combined extracts)

Fraction collectorRate of collection 0.1 min/tubeNumber of fractions 50Start time 0.6 minEnd time 5.6 min

Diode array detectorWavelength 280nmSlit 4 nmData rate 40 HzFlow cell 10 mm

SoftwareOpenLAB CDS, ChemStation Edition (Agilent Technologies, Mulgrave, Australia)Wolfram Mathematica (version 9) - (Wolfram Research, Champaign, IL, USA)

ChromatograhyA 1260 Agilent Infinity HPLC with 1260 Infinity autosampler, thermostatted column compartment, diode array detector, a 1290 binary pump and a Gilson analytical-scale fraction collector was used for this study. Table 1 lists the chromatographic conditions used.

Table 1. HPLC set up conditions

Result and DiscussionFigure 2 shows an initial one-dimensional separation of the mushroom extract using a C18 Luna column with a gradient protocol starting from 5% methanol at 0 mins, to 100% methanol at 30 mins. Five classes of interest are marked as follows: A-sugars, B-organic acids, C-amino acids, D-phenolics and E-fatty acids. The significant overlapping of the sugars, organic acids and amino acids in the first 10 mins of the run makes it difficult for identification and quantification of metabolites, even when an LC-MS was used. A closer look at the separation from 21 to 27 mins (inset panel) also shows that the peaks are not baseline resolved.

Figure 2. One-dimensional separation of mushroom extract on a C18 column (150 mm x 4.6 μm x 5 μm)

One of the most important factors to consider for 2DLC method development is the phases of the two columns (dimensions). To ensure high-resolution separation the columns should consist of different (and unrelated) phases so that the peaks are spread across the maximum separation area. A selectivity study of four stationary phases (CN, NH2, PFP and C18) using the mushroom extract was therefore undertaken and the results are shown in Figure 3.

The combination of CN and the C18 were found to demonstrate the greatest the separation of peaks in this case (although alternative column combinations may be necessary for different sample types). Moreover the chromatogram profiles are different, even though the CN was run under reverse phase conditions the CN groups offer sufficient polar characteristics to alter the interactions between the stationary phase and the mobile phase compared to a C18 based column.

Two common challenges faced by 2DLC users are that the total analysis run times are long; and the fact that samples often become highly diluted in the second column. To counteract this, both dimension run times were adjusted so that the run time was as short as possible and the columns were purposely overloaded with injection volume of 100 μL. This results in the unsymmetrical peak broadening seen in figure 4 (below).

Figure 3. Selectivity study of 4 Luna columns with stationary phases of; A) CN, B) NH2, C) PFP and D) C18. All columns had dimensions of 150 mm x 4.6 mm x 5 µm. The mobile phase in this part of the study started at 95% H2O: 5% methanol through to 100% methanol by 20 mins, which was then held for 10 mins

Figure 4. One-dimensional chromatogram of the mushroom extract using a CN column. This data is the first dimension (x-axis) in figures 6 and 7

Although the separation in figure 4 at first appears poor, the, peak separation and total capacity is later enhanced due to use of the second (C18) column which provided a second dimension of separation.

We chose to use a Kinetex C18 with core-shell particles instead of the Luna C18 phase (which has fully porous particles) for the second dimension separation since this resulted in less band broadening and provided better resolution, sensitivity and peak capacity.

The next step is to take the data from each fraction and combine to a data matrix with time in column 1 as the x dimension and time in column 2 in the y dimension and the intensity or response at each time point as the z value (either colour or height) [3]. The resulting 2DLC offline separations are shown in Figures 6 and 7 as a 2D contour plot and a 3D intensity plot, each created using Mathmatica. The separation of a total of 221 components was achieved.

Figure 5. One-dimensional chromatogram of the mushroom extract using a Kinetex C18 column. This data is the second dimension (y-axis) in figures 6 and 7

Figure 6. Two-dimensional contour plot using data from separation run using CN and Kinetex C18 as first and second dimensions respectively.

CONCLUSIONThis method detailed here demonstrates that a one-dimensional Agilent system coupled with a fraction collector can be used for the non-automated, two-dimensional comprehensive analysis of complex matrices. Although the total analysis time of the non-automated approach is longer than a fully automated separation, a greater peak capacity is obtained since the length of second dimension is not restricted, meaning that longer columns can be used in both dimensions. The greatest separation of peaks was obtained using reversed phase HPLC with CN and C18 based columns for the first and second dimensions respectively. The method was used to profile metabolites in mushrooms could easily be adapted and applied to other complex sample matrices.

REFERENCES1. Jones, O.A.H. and H.M. Hügel, Bridging the Gap: Basic Metabolomics Methods for Natural Product Chemistry, in Metabolomics Tools for Natural Product Discovery, U. Roessner, Dias, D.A., Editor 2013, Humana Press: London, UK. p. 245-266.

2. Le Belle, J., N. Harris, S. Williams, and K. Bhakoo, A Comparison Of Cell And Tissue Extraction Techniques Using High- Resolution 1H-NMR Spectroscopy. NMR in Biomedicine, 2002. 15: p. 37-44.

3. Stevenson, P.G., M. Mnatsakanyan, G. Guiochon, and R.A. Shalliker, Peak Picking And The Assessment Of Separation Performance In Two-Dimensional High Performance Liquid Chromatography. Analyst, 2010. 135: p. 1541-50.

Figure 7. Data from figure 6 plotted as a three dimensional chromatogram