Basic Techniques of Preparative Liquid Chromatography and ... · Basic Techniques of Preparative...

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Technical Report vol.31 Basic Techniques of Preparative Liquid Chromatography and Approaches to Efficiency Improvement (1) C190-E127 1. Roles of a Preparative System A preparative system is a liquid chromatograph (LC) that is used to recover high purity components from some type of liquid extract obtained from either a synthesis reaction or from a natural substance by separating and purifying the target compounds. Obtaining these target compounds at high purity enables their structural analysis, permits evaluation and analysis of their various functions, and allows their subsequent processing to be conducted more reliably. Since a preparative system must separate the target components from the coexisting substances in a sample, the basic system configuration comprises a solvent delivery pump, sample injector, column and detector, as in a typical LC system. Added to these, however, is a mechanism for collecting fractions consisting of the target substances, thereby completing the basic configuration of a preparative system. In an extremely simple preparative system, manual injection and manual fractionation techniques are conducted using a manual injector as the sample injector, and a manual flow line switching valve as the fractionation device (see Fig. 1). The objectives of a typical LC system are to conduct quantitative and qualitative analysis, but a preparative system is used more for so-called "pretreatment" with the objective of actually obtaining the necessary compounds for evaluation and analysis, as well as for subsequent processing. Therefore, it is important that target compounds be obtained quickly and at high purity, and similarly as with a typical pretreatment process, productivity is the key point in a preparative system. To ensure that the intended evaluation and analysis operations can be started as soon as possible following the preparative process to achieve the highest possible productivity, an appropriate system must be selected based on: (1) the quantity of compounds required to conduct the intended evaluation and analysis, and (2) the detection principle that can most effectively ensure collection of the target compounds. With respect to (1), if the required quantity can presently be collected in five runs of fractionation using a 20 mm inner diameter (I. D.) column, introduction of a 50 mm column and a system that can accommodate that column would allow that same quantity to be collected in just a single run, making it possible to greatly increase the throughput of the target compound evaluation and analysis, and all subsequent processing. 2. Improving Fractionation Productivity Fig. 1 Simple Preparative System Pump Manual injector Column Detector Manual flow line switching valve (fractionation) Fraction Waste liquid

Transcript of Basic Techniques of Preparative Liquid Chromatography and ... · Basic Techniques of Preparative...

Technical Repor t vol .31

Basic Techniques of Preparative Liquid Chromatography and Approaches to Efficiency Improvement (1)

C190-E127

1. Roles of a Preparative SystemA preparative system is a liquid chromatograph (LC) that is used to recover high purity components from some type of liquid extract obtained from either a synthesis reaction or from a natural substance by separating and purifying the target compounds. Obtaining these target compounds at high purity enables their structural analysis, permits evaluation and analysis of their various functions, and allows their subsequent processing to be conducted more reliably. Since a preparative system must separate the target components from the coexisting substances in a sample, the basic system configuration comprises a solvent delivery pump, sample injector, column and detector, as in a typical LC system. Added to these, however, is a mechanism for collecting fractions consisting of the target substances, thereby completing the basic configuration of a preparative system. In an extremely simple preparative system, manual injection and manual fractionation techniques are conducted using a manual injector as the sample injector, and a manual flow line switching valve as the fractionation device (see Fig. 1).

The objectives of a typical LC system are to conduct quantitative and qualitative analysis, but a preparative system is used more for so-called "pretreatment" with the objective of actually obtaining the necessary compounds for evaluation and analysis, as well as for subsequent processing. Therefore, it is important that target compounds be obtained quickly and at high purity, and similarly as with a typical pretreatment process, productivity is the key point in a preparative system. To ensure that the intended evaluation and analysis operations can be started as soon as possible following the preparative process to achieve the highest possible productivity, an appropriate system must be selected based on:(1) the quantity of compounds required to conduct the intended evaluation and analysis, and(2) the detection principle that can most effectively ensure collection of the target compounds.With respect to (1), if the required quantity can presently be collected in five runs of fractionation using a 20 mm inner diameter (I. D.) column, introduction of a 50 mm column and a system that can accommodate that column would allow that same quantity to be collected in just a single run, making it possible to greatly increase the throughput of the target compound evaluation and analysis, and all subsequent processing.

2. Improving Fractionation Productivity

Fig. 1 Simple Preparative System

PumpManual injector

ColumnDetector

Manual flow line switching valve (fractionation)

Fraction

Waste liquid

Chromatogram (MS)

Chromatogram (LC)

Sample rack

Fractions

Regarding detector selection of item (2) above, if the target compound has no optical absorbance, or the coexisting impurities in the sample solvent have no optical absorbance, detectors that are based on principles other than optical absorbance detection, such as the ELSD (evaporative light scattering detector) and MS (mass spectrometer), can be used as supplementary detectors to improve purification efficiency, and thereby improve productivity. Fig. 3 shows examples of detectors that can be used to supplement absorbance detectors, while Fig. 4 shows a possible configuration in which an ELSD or MS is used as an additional detector in the simple preparative system of Fig. 1. Since the ELSD and MS detectors in principle rely on nebulization of the column eluate inside the detector, the target substances emerging from these detectors cannot be recovered. To resolve this problem, most of the column eluate is directed to the fraction collector, and the remaining micro volume is introduced into the ELSD or MS. To accomplish this, an active splitter (APV: Automated Proportioning Valve) is used to split the flow line. In addition, if the volume of eluate to be introduced into the ELSD or MS detector is ultra small, a make-up pump is used to augment the mobile phase flow and maintain steady detection by the ELSD or MS.As described in Technical Report No. 15 "Selecting Detectors for Compounds with No Optical Absorbance", nonvolatile buffer solution cannot be used as a mobile phase with ELSD and MS detectors. However, since a preparative system is used with the aim of conducting subsequent evaluation and analysis, and further processing, the use of nonvolatile buffer solutions is often avoided, making ELSD and MS detectors highly compatible with preparative systems. For example, instead of the phosphate buffer solution often used in LC analysis, formic acid, acetic acid, or trifluoroacetic acid, etc. are used as mobile phases in preparative LC. These are generally used as mobile phases with ELSD and MS detector.

(Note)

The APV is an automatic flow line switching unit that automatically splits the

flow. Since it provides split flow ratios as high as 1,000 : 1 or even 10,000 : 1,

very high quantities of target compounds can be collected. It is possible to use

a restrictor tube instead of the APV, but with the restrictor tube, the split ratio

is maintained according to the pressure ratio. Therefore, the split ratio will

change whenever there is a change made to the flow rate or to the

composition of the mobile phase or makeup solution. For that reason, the

restrictor tube must be modified according to the analytical conditions in order

to maintain the split ratio. On the other hand, that sort of task is unnecessary

with an APV, because flow line switching is conducted according a specified

parameter setting.

When using a high split ratio of 1,000 : 1 or greater, the flow rate to the ELDS

or MS becomes extremely small, in the order of a few μL/min. The makeup

pump serves to supplement the flow to the detector due to the insufficient

mobile phase in the flow line.

2

LC-20AT Pump

LC-6AD Pump

LC-8A Pump

Refractive Index Detector RID-10A

Makeup pump (Note)

PumpManual injector

Column

APV (Note) ELSD or MS

UV detector Waste liquid

Fraction collector

Fraction

Waste liquid

Evaporative Light Scattering Detector

ELSD-LTII

Mass Spectrometer LCMS-2020

Fig. 2 Column Scale and Corresponding Solvent Delivery Pump

Fig. 3 Detectors for Supplementing Optical Absorbance Detectors

Fig. 4 Example of ELSD or MS Added to Simple Preparative System (Fig. 1)

3 mg3 mg 20 mg20 mg 300 mg 2 g20 mg 300 mg 2 g

2 mmI.D.

0.001 0.01 0.1 1 10 1000.001 0.01 0.1 1 10 100

5 mmI.D. 20 mmI.D. 50 mmI.D.

LC-20AT

LC-6AD

LC-8A

10 mL/min10 mL/min

20 mL/min20 mL/min

150 mL/min

Maximum load guide (1 injection, 25 cm long column)

Flow Rate mL/min

Semi micro Analytical Semi analytical PreparativeSemi micro Analytical Semi analytical Preparative

Flow Rate mL/min

Semi micro Analytical Semi analytical Preparative

3

3. Determining Fraction Quantity and Preparative SystemOnce the total amount of fraction necessary for evaluation and analysis of the target compounds and subsequent processing is calculated, and the detector has been selected, the next step is to determine what scale of fractionation is required (see Fig. 2). The amount of sample that can be injected at one time (load with respect to column) to maximize productivity must also be determined. Once the maximum sample injection volume is known, the number of required fractionation runs can be calculated based on the target fraction quantity. This allows determination of the optimum preparative system based on time (total time to collect the required fraction) and cost (purchase price of preparative system and column, and solvent expense).This type of examination can be conducted efficiently by first using a general purpose LC (conventional LC). The process is referred to as "scale-up" from an analytical system to a preparative system, and follows the procedure shown in Fig. 5.

4. Calculating Absolute Injection QuantityIf the concentration of the fractionation target compound in the sample is known, the absolute injection quantity of the fractionation target compound can be obtained from this concentration and the sample injection volume. An example of this is shown in Table 1, in which the absolute injection quantity is indicated for the cases where the concentrations in the sample range from 0.01 mg/L to 10,000 mg/L, and the sample injection volumes are 10 μL and 100 μL, respectively.Referring to this table, if the concentration of the target fraction compound is 100 mg/L, a sample injection volume of 100 μL is indicated in order to obtain a 10 μg fraction.

5. Calculating Maximum Sample LoadThe advantage of having a large-sized column and a preparative system that supports the column is the ability to conduct fractionation at various scales. However, due to the relatively high acquisition and operational costs associated with such a system, lab operators will often choose the approach of conducting more repetitive fractionation using a compact column and smaller scale system. The strategy for raising productivity in this scenario is to reduce the number of fractionation repetitions, and increase the sample injection volume per run (column load) as much as possible to obtain the largest fraction possible of the target compound per sample injection. In preparative LC, unlike the situation in typical LC analysis, there is no need to be concerned about the sharpness or shape of a target

peak, and furthermore, since there is no need to conduct quantitation, there is no need to worry about detector signal saturation as long as peak separation is adequately maintained. As shown in Fig. 6, as the sample injection volume per injection (column load) is increased, a chromatographic phenomenon known as peak fronting is observed, indicating column overload, that is, the appropriate load for that column has been exceeded. However, as long as separation is maintained between neighboring peaks, fractionation can be conducted.

<<Analytical Scale>> <<Preparative Scale>>

Fig. 5 Scaling Up Procedure

Fig. 6 Changes in Peak Shape Due to Changes in Sample Load

ColumnMobile phaseFlow rateSample

: Shim-pack PREP-ODS(L) 50 mm I.D. x 250 mm L.: water/methanol = 2/3: 100 mL/min: Phenol, 2-naphthol

Table 1 Absolute Injection Quantity

3 mg 20 mg 300 mg 2 g

0.001 0.01 0.1 1 10 100

10 mL/min

20 mL/min

Flow Rate mL/min

Semi micro Analytical Semi analytical PreparativeSemi micro Analytical Semi analytical Preparative

Yes

Yes

No

No

2 g

600 mg

200 mg

60 mg

20 mg

0 4 8 12 16 20 24 28 min

Create separation conditions.

Is peak separation secured?

Fix conditions based on

analytical scale.

After scale-up, observe elution pattern using

preparative column.

Determine fractionation conditions.

Verify purity of fractionation

components.

Execute repeated fractionation.

Concentration in Sample Solvent

(mg/L)

0.011

10010,000

0.0010.01

1100

0.0010.110

1,000

Absolute Injection Quantity (μg)

With 10 μL Sample Injection With 100 μL Sample Injection

This shows the importance of knowing the maximum sample load on the column in setting up fractionation for high productivity. In this examination, conventional 4.6 mm and 6 mm I. D. columns normally employed in typical analysis are used in these trial analyses to obtain an approximate guideline. Fig. 7 shows an example of maximum sample load investigation using a conventional 250 mm (L) x 4.6 mm (I. D.) ODS column. Here, in which peak 2 (benzene) is the target fractionation substance, there are no problems with respect to peak shape or separation in chromatograms A and B. In chromatogram C, despite the evidence of some peak distortion, there is sufficient resolution to use this injection volume for fraction collection. In chromatogram D, however, there would be a problem with fraction purity because there is no separation from peak 3. From this it is clear that conventional-scale fraction collection is possible with a sample injection volume of 50 μL, which is equivalent to an absolute injection quantity of 25 mg.Once the maximum sample load is determined for the conventional scale, examination of the maximum sample load is conducted for the preparative scale according to 9. Scale-Up to Preparative System. It should be noted that the injection volume was increased while the sample concentration remained fixed in this example, but it is also possible to increase the sample concentration while keeping the injection volume constant.

6. Effect of Packing Material Particle Size on ResolutionSelecting a column with very small-diameter packing material particles generally provides good separation, however, increasing the sample load will eventually negate any changes in resolution due to differences in packing material particle size, as can be seen in Fig. 8.Preparative columns with packing material consisting of small-diameter particles are generally expensive, and further, since pressure is higher during solvent delivery, the merits of sample load and cost should be carefully considered in the selection of such a column.

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Injection Quantity (g/cm2)

Res

olut

ion

Fig. 8 Effect of Packing Material Particle Size on Resolution

Fig. 7 Examination of Maximum Load with Conventional Column

<<A>>Injection Volume 10 μL

Benzene 5 mg

0 2

2

1

3

4

4 6 8

<<B>>Injection Volume 20 μL

Benzene 5 mg

2

1

3

4

0 2 4 6 8

<<C>>Injection Volume 50 μL

Benzene 25 mg

2

1

3

4

0 2 4 6 8

<<D>>Injection Volume 100 μL

Benzene 50 mg

21

4

0 2 4 6 8

25

20

15

10

5

10-6 10-5 10-4 10-3 10-2 10-1

ColumnMobile phaseFlow rateSample

: Shim-pack PREP-ODS: water/methanol = 2/3: 30.2 mm/min: phenol, 2-naphthol

5

7. Effect of Sample SolventWhen using a larger sample injection volume to increase the sample load, it is also necessary to examine the effect on separation of the sample solvent components. When the sample solvent elution strength is greater than that of the mobile phase, distortion of the peak shape will be more apparent as greater injection volumes are used. Fig. 9 shows the changes in theoretical plate number when injecting different volumes as well as different types of sample solvent in reversed-phase analysis. The mobile phase consists of water/methanol = 3/7. The absolute injection quantity of the target compound was kept constant for every injection. When using water/methanol = 7/3 and methanol as sample solvents, the sample solvent elution strength is greater than that of the respective mobile phases, so as the injection volume increased, an obvious degradation of peak theoretical plate number occurred. Fig. 10 shows the changes in peak shape with different injection volumes using methanol as the sample solvent with the mobile phase conditions shown in Fig. 9.

As can be seen from these results, it is not just the sample load on the column, but the injection volume as well that can influence the peak shape. This phenomenon occurs due to the influences of the sample solvent and mobile phase on the target substance elution behavior when using large injection volumes.The peak distortion seen in Fig. 10 is illustrated graphically in Fig. 11(a). When the elution strength of the sample solvent is greater than that of the mobile phase, it forms a separate band of high elution strength mobile phase when larger injection volumes are used. A portion of the solute in the sample moves continuously through the column while being pulled along with this sample solvent band, and as a result, peak shape distortion occurs. As the sample injection volume increases, this band becomes wider, and since the quantity of substances included in the band increases, peak distortion becomes more accentuated. This phenomenon can be inhibited by modifying the sample solvent so that its elution strength is either equivalent to or weaker than that of the mobile phase. Changing to a sample solvent with less elution strength should result in sharper peaks and improved resolution because the solute is concentrated in the vicinity where it is introduced into the column (see Fig. 11(b)).

Injection Volume (μL)

The

oret

ical

Pla

te N

umbe

r (x

103

)

Fig. 10 Injection Volume and Peak Shape with High Elution Strength Sample Solvent Using Constant Absolute Injection Quantity

Fig. 9 Effects of Sample Solvent and Sample Injection Volume

Sample solvent: methanol

Elution has begun

Sample

Sample solvent: methanol

(a) When elution strength of sample solvent is high

(b) When elution strength of sample solvent is low

Target substance

Sample solvent: water

Target substance

Sample injection

Liquid flow Mobile phase: water/methanol

Sample

Sample injection

Liquid flow Mobile phase: water/methanol

Fig. 11 Effect of Sample Solvent

ColumnMobile phaseFlow rateSample

: Shim-pack CLC-ODS 6 mmI.D. x 150 mmL: water/methanol = 3/7: 1 mL/min: caffeine 10 μg

15

10

5

01 10 100 1,000

WaterWater/methanol = 7/3Methanol

0 2 4 6 8 min

0.1 mg/mL x 100 μL

0.2 mg/mL x 50 μL

1.0 mg/mL x 10 μL

8. Influence of Column TemperatureSometimes a column oven is used with a 20 mm I. D. semi-preparative column, but in general, a column oven is not used in preparative LC. That is because peak distortion sometimes occurs when a column oven is used. Fig. 12 shows chromatograms obtained using a 20 mm I. D. column with a 20 mL/min solvent delivery flow rate. Good peak shapes are seen at near-ambient temperature, but as the oven temperature is raised, the peaks become distorted. When using a column with a large internal diameter, the heat of the oven is not sufficiently conducted to the axial region of the column, thereby generating a temperature gradient across the column cross section. Therefore, the solute movement rate at the axial and radial regions of the column differs, causing a widening of the band of eluting substances. This phenomenon becomes more pronounced when 1) there is a large difference between the mobile phase temperature (ambient temperature) and oven temperature, 2) the column internal diameter is large, and 3) the solvent delivery flow rate is high. If a column oven is to be used, techniques that are used to alleviate temperature gradient problems include connecting stainless steel tubing before the column inside the oven to warm (preheat) the mobile phase before it reaches the oven.

9. Scale-Up to Preparative SystemAfter examining the conditions that were used with the conventional-size column, the next step is to scale up from the analysis level to the preparative level. To smooth this transition, a preparative column is selected that uses the same packing material as that which was used in the analytical column. If the packing material is the same, the mobile phase flow rate and sample injection volume need only be increased according to the analytical column and preparative column cross-sectional area ratio to obtain nearly the same chromatographic pattern (see Table 2).An example in which the scale-up was conducted using the same packing material is shown in Fig. 13. Here, the 4.6 mm I. D. column used for the analytical scale was exchanged for a 20 mm I. D. column for the preparative scale. Since the cross-sectional area of the 20 mm I. D. column is about 19 times that of the 4.6 mm I. D. column, the solvent delivery flow rate was scaled up from 0.8 mL/min to 15 mL/min, while the sample injection volume was scaled up from 50 μL to 1 mL. As a result, nearly equivalent chromatographic patterns were obtained.

10. Checking Purity of Fractionated SubstancesAfter confirming the scale-up by comparing the chromatograms, the collected fraction solution is re-injected to verify the purity. Here, if the target purity is not achieved because of insufficient separation, the sample injection volume and column length, etc. must be reexamined, and in the case of gradient analysis, the gradient conditions must also be reexamined. In the event that high sensitivity is required while using a small injection volume in this purity confirmation analysis, use of an analytical system for verification rather than a preparative system is suitable. Thus, when considering scale-up from an analytical system to a preparative system, it is important to realize that a system which is applicable to both analytical and preparative LC effectively allows the preparative part of the system to be used together with the analytical part of the system, thereby improving productivity.

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Fig. 13 Results of Scale-Up from Analytical to Preparative

Fig. 12 Effect of Column Oven Temperature on Semi-Prep Column

<<Analytical Scale>> <<Preparative Scale>>Column I. D.Solvent delivery flow rateSample injection volume

: 4.6 mm: 0.8 mL/min: 50 μL

Column I. D.Solvent delivery flow rateSample injection volume

: 20 mm: 15 mL/min: 1 μL

0

26°C

40°C

50°C

5 10 15 min

0 2

1

2

4

3

4 6 8

1

2

4

3

0 2 4 6 8

Table 2 Relationship Between Column Cross-sectional Area and Flow Rate/Injection Volume

Column

Internal Diameter

(mm)

Cross-Sectional

Area (mm2)

Cross-Sectional Area Ratio Based

on "1" as 4.6 mm I. D. Cross-sectional Area

Flow Rate Based on "1"

as 4.6 mm I. D. Flow Rate (mL/min)

Injection Volume Based on "10" as 4.6 mm I. D.

Injection Volume (μL)

4.6

20

50

17

314

1,963

1

19

115

10

190

1,150

1

19

115

7

11. Analytical/Preparative Automatic Switching SystemA system that can handle both analytical and preparative scale LC chromatography can effectively and easily be used to check the preparative conditions. Such a system is called an analytical/preparative automatic switching system. Fig. 14 shows a photograph of one such type of this system, and its associated flow line diagram. Here, LC-8A pump units are used for solvent delivery. Fig. 15 shows a scale-up example using this system.

Using such an analytical/preparative automatic switching system not only facilitates verification of separation conditions and sample load using a conventional column, but also makes possible fraction purity verification following scale-up, all using a single system. This translates into greatly improved productivity for preparative LC.

Analytical mixer

Pump

Pump

Flow line switching valve

Analytical column

Detector

Fraction collector

Waste liquid

Preparative mixer

Preparative columnPreparative

injector

Analytical injector

Fig. 14 Analytical/Preparative Automatic Switching System

Fig. 15 Scale-Up Example Using Analytical/Preparative Automatic Switching System

Column

Mobile phaseFlow rateSample

: Shim-pack PREP-ODS(H)kit A)250 mmL. x 4.6 mmI.D., 5μm B)250 mmL. x 20 mmI.D., 5μm: 10 mmol/L Phosphoric acid (sodium) buffer solution (pH 2.6) / methanol =1/9 (v/v): A)0.8mL/min B)15mL/min: A)5μL, B)100μL

1. 2. 3. 4. 5. 6. 8. 9.

10. 11. 12. 14. 15. 16.

Peak Substances

phthalic acidcaffeic acidsalicylic acidbenzoic acid2-naphtholbenzeneisopropyl benzoaten-propyl benzoatenaphthalenebiphenylpentyl benzoatephenanthreneanthracenefluoranthene

0 4 8 122 6 10 14 0 4 8 122 6 10 14

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In preparative LC, unless meticulous care is taken to track the sample and chromatogram associated with fractionation, as well as the chromatographic peaks and vials in which those peaks (target substance fractions) are collected, not only can the collected compounds be wasted, but the subsequent processes can be severely compromised. When this sample tracking can be simply and securely achieved, the productivity required for preparative analysis will be greatly improved.The software that achieves such productivity improvement is Open Solution.

12. Simplifying Verif ication of Preparative Results

With Open Solution, the fractionation results can be perceived instantly and reliably because the vial rack diagram, fractionation results and fraction vials are all linked (see Fig. 16). Moreover, even more complex operations can be conducted using Open Solution, as for example when conducting re-injection of the fraction solution for verification of purity. The Gilson 215 liquid handler can be used to re-inject the fraction solution and the results can be viewed visually, greatly improving preparative productivity.

*The above Internet Explorer window displays an example of results obtained using two detectors simultaneously, a mass spectrometer and a photodiode array detector.

References·LCtalk VOL. 68/69: in the LAB "Establishing Preparative Conditions by Scaling-Up" (Japanese)·Technical Report vol. 6: Principles and Practical Applications of Shimadzu's ELSD-LTII Evaporative Light Scattering Detector·Technical Report vol. 15: Selecting Detectors for Compounds with No Optical Absorbance·Technical Report vol. 21: Reducing Analysis Limitations by Using Multiple Detectors with Ultra Fast LC·Technical Report vol. 29: Open Solution: Open Access Environment by Web Browser·Technical Report vol. 30: Improved R&D Efficiency in Compound Synthesis, Confirmation and Purification

Fig. 16 Mutually Linked Fractionation Results, Vial Rack and Fractions Using Open Solution

Magnification of chromatograms and spectra can be freely adjusted.

When a more detailed report is required, just click on this icon to output a report using Solutions series format, applying advanced functionality to report output.

Click on a peak in the mass chromatogram to highlight the corresponding fraction vial.

Click on a target vial to display the corresponding results (fraction vial, chromatograms, spectra, peak table).

Click on a fraction vial to highlight the corresponding peak.

Click on a peak in the PDA (photodiode array) detector data to display its spectrum.

Chromatogram (MS)

Chromatogram (LC)

Sample rack

Fractions

Chromatogram (MS)

Chromatogram (LC)

Mass spectrum

Sample rack

Fractions

UV Spectrum