Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products?Rainer Bauder1, Frank Steiner2, Michael Heidorn2, Markus Martin2, Fraser McLeod2

1Thermo Fisher Scientific, Chelmsford, MA; 2Thermo Fisher Scientific, Germering, Germany

2 Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products?

Table 2: Chromatography methods overview and peak capacity gains, black cohosh

Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products? Rainer Bauder1, Frank Steiner2, Michael Heidorn2, Markus Martin2, Fraser McLeod2 1Thermo Fisher Scientific, Chelmsford, MA; 2Thermo Fisher Scientific, Germering, Germany

Conclusion   Combining individual 150 mm or 250 mm UHPLC columns to achieve extended

column length and column volumes, is a feasible approach to boost separation of analytes found in complex samples such as botanical supplements.

  The approach is applicable to UV/VIS, charged aerosol, or mass spectrometry detection.

  Charged aerosol detection is a near universal responding detection technique to quantify components without chromophores and therefore an excellent addition to UV/VIS or MS detection.

  Improved resolution now enables the measurement of low abundant impurities that previously remained hidden by the more abundant analytes.

  Shallow gradients are able to provide improved separation for a wider range of samples and botanicals.

  The concept can also be applied to early drug discovery or drug development.

  Natural products from botanical sources can be better investigated and “fingerprinted” for adulteration testing and quality control. The concepts enable an in-depth assaying for relatively unknown samples and matrices.

References 1.  T. W. D. Chan; P. P. H. But; S. W. Cheng; I. M. Y. Kwok; F. W. Lau; and H. X. Xu;

"Differentiation and Authentication of Panax ginseng, Panax quin-quefolius, and Ginseng Products by Using HPCL/MS" Analytical Chemistry, 2000, 72 (10), 2329–2329.

2.  Avula B; Wang YH; Smillie TJ; Khan IA; "Quantitative determination of triterpenoids and formononetin in rhizomes of black cohosh (Actaea racemosa) and dietary supplements by using UPLC-UV/ELS detection and identification by UPLC-MS". Planta Med. 75 (4): 381–6. doi:10.1055/s-0028-1088384. PMID 19061153 (March 2009).

Overview Purpose: Evaluation of high peak capacity UHPLC setups and alternative, near universal detection technologies to improve separation and analysis of complex samples from natural sources.

Methods: Various serial arrangements of long, 2 µm particle UHPLC columns were used to investigate resolution improvements of selected botanical extracts.

Results: High efficiency UHPLC columns in 150 mm length (2 µm particles) already provide very high resolution chromatography. The use of longer UHPLC columns or the combination of 2 × 250 mm 2 µm C18 columns almost doubles peak capacities even for very complex samples. The improved resolution benefits component identification and isolation for further, in-depth investigation. This approach is a widely applicable strategy and is complemented by the near universal response of the Thermo Scientific Dionex Corona ultra RS detector.

Introduction

Natural product phytochemicals derived from botanical sources, are undergoing a revived interest in targeted drug development. In particular, traditional medicines from various geographies provide a truly global offering for promising leads. However, botanical samples often exhibit a significant complexity and many of their ingredients do not contain a chromophore or cannot readily be ionized. Hence, their analysis can be extremely challenging, both chromatographically and due to the limited ability to use UV absorbance and mass spectrometry detection.

High resolution UHPLC, using long columns packed with small particles and shallow gradients, is a very practical way to boost peak capacity. With the subsequent increase in resolution, small sized peaks eluting next to much larger ones can be more accurately integrated; thus characterization quality of complex mixtures is improved. This can be critical when a non-selective detector like the charged aerosol detector (Corona™ ultra RS™) is used. With the selection of herbal medicines taken from around the world, the effectiveness of using very long UHPLC columns with a wide range of detection techniques (charged aerosol detection, UV/VIS) is evaluated in this work.

Ginseng is any one of eleven species of slow-growing perennial plants with fleshy roots, belonging to the genus Panax of the family Araliaceae. Ginsenosides found at high abundance in the root are the purported active compounds that can be used to distinguish different Panax species. Herbal companies who follow Good Manufacturing Practices (GMP) regularly test for the quality, potency, and species authentication of their herbs. One published study found HPLC was especially useful in the differentiation and authentication of Panax ginseng from Panax quinquefolius due to the unambiguous distinction of slightly varying isotypes of ginsenoside compounds.1

Actaea racemosa (black cohosh, black bugbane, black snakeroot, fairy candle) is a plant from the buttercup family. Roots and rhizomes of black cohosh have long been used medicinally by Native Americans to treat gynecological and other disorders, including sore throats, kidney problems, and depression. Triterpenoids and formononetin in rhizomes of black cohosh have been previously studied and quantified using UHPLC and MS techniques.2

The milk thistle, silybum marianum, is a thistle of the genus Silybum Adans., a flowering plant of the daisy family (Asteraceae). They are native to the Mediterranean regions of Europe, North Africa and the Middle East. The name "milk thistle" derives from two features of the leaves; they are mottled with splashes of white and they contain a milky sap. Research is being undertaken on the physiological effects, potential therapeutic properties, and possible medical uses of milk thistle.

The above described, over-the-counter and readily available preparations have been selected to demonstrate the very practical and easy approach for boosting peak capacities and column efficiencies beyond commonly available UHPLC column formats. They also contain, if not proven but suggested, pharmaceutically active and valuable components which are likely to be studied further in the search of new active entities.

Methods

Sample Preparation

Herbal over-the-counter preparations of Korean white ginseng and black cohosh were extracted with methanol. About 500 mg of the capsules’ contents were sonicated for 15 min in 10 mL methanol (HPLC gradient grade). The milk thistle extract was prepared with the same approach using ca. 50 mg powdered content of the capsule in 5 mL methanol. The raw extracts were filtered through 0.45 µm syringe filters prior to transfer into the sample vials.

Liquid Chromatography System: Thermo Scientific Dionex UltiMate 3000 RSLC system

equipped with: LPG-3400XRS, DGP-3600RS, WPS-3000(X)RS, TCC-3000RS, DAD-3000RS, Corona ultra RS

Columns: Thermo Scientific Acclaim RSLC120 C18, 2.1mm ID, 2.2 µm particle, 150 mm, 250 mm and in combination

Mobile Phase A: 0.1% Formic acid in HPLC gradient grade water Mobile Phase B: 0.1% Formic acid in HPLC gradient grade methanol Flow Rate 0.40 mL/min Gradient Conditions: ginseng, cohosh => 45% B hold 3.0 min, 100% B in

20 min hold for 5.0 min, return and equilibrate for 7.0 min, other gradient times as indicated milk thistle => 17%B hold 3.0min, 43%B in 23.0min, 70% B in 0.1 min, hold for 5.0 min, 5.0 min to equilibrate

Injection Volume: 3–5 µL Corona ultra RS settings: Filter: 3 Nebulizer Temp On at 25 °C

Power Function Value 1.0

Mass Spectrometry

Mass spectrometry was not used for this evaluation.

Data Analysis

Thermo Scientific Dionex Chromeleon 7.1 SR.1 Chromatography Data System (CDS) was used for all data processing.

Results

Peak Capacity Improvements When Increasing Column Length While Maintaining Relative Gradient Time

Further improvement of resolution, even when having already applied 2 µm or sub 2 µm particle columns, is often only possible by adding column length. This approach has multiple benefits. First, the method does not need to be redeveloped as gradient times and slopes change relative to the added column length. For example, Table 1 shows, that the peak capacity for the ginseng extract jumps from 142 (40 °C, 150 mm) to 335 (75° C, 500 mm). Even when maintaining the original column temperature, the extension of the original 150 mm column length to 400 mm almost doubles peak capacity. Table 2 shows the results for black cohosh extract. Comparable efficiency improvements would require to reduce the particle size by half, which in this example would demand a 1µm particle column.

Second, with increased column volume and capacity, more sample and sample matrix are tolerated by the column. Low abundant peaks will be resolved better from prominent main peaks as can be seen in figures 2, 4, and 5. The time axis for the displayed traces are not to scale and allow a good, visual comparison. Please refer to tables 1 and 2 for gradient times and overall runtimes (150 mm and 500 mm column length for Figures 1-5).

Using peak capacity as the measure for separation performance of a given LC or UHPLC method is a very practical way to assess achievable resolution. The theoretical plate count for single peaks can only imperfectly describe the overall separation success. For this reason, the peak capacity was calculated by dividing the active gradient time with the averaged base peak width of all peaks found. To maintain comparative peak identification results, constant peak detection parameters were chosen and applied to all injections of the same extract.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO70153_E 06/12S

FIGURE 1: Overlay Korean white ginseng, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150 mm = 20 min

FIGURE 5: Zoomed overlay milk thistle extract, UV-detection at 288 nm tgradient 500mm = 90 min, tgradient 150mm = 23 min

Improved peak capacity and separation as observed for ginseng was also found for black cohosh. This is clearly illustrated in the zoomed overlays in Figures 2 and 4.

Although the use of a longer column required a simple adjustment in gradient time and column temperature, no additional method development was necessary. This straight- forward approach can consequently very easily be introduced as a standard operating procedure for new projects and (unknown) samples (matrices).

For mass spectrometry, the increasing peak width as shown in tables 1 and 2, have also very practical and beneficial impacts. While chromatographic resolution overall is improved, there is also more time to acquire data points. This improves the likelihood to measure and detect minor peaks. With fast collecting fraction collectors (2D-offline methods or 2D-comprehensive LC methods), trace and low abundant components can be further enriched and investigated.

Core enhanced particle UHPLC columns offer another way to improve peak sharpness and resolution. The chromatographer benefits from the faster mass transfer between mobile phase and stationary phase. Typically, this has to be traded off with reduced backpressure stability or reduced column loading capacity.

FIGURE 3: Overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

Table 1: Chromatography methods overview and peak capacity gains, Korean white ginseng

FIGURE 4: Zoomed overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

FIGURE 2: Zoomed overlay Korean white ginseng, Corona ultra RS tgradient 500mm = 66min, tgradient 150mm = 20min

In the case of the milk thistle extract, the sample was much less complex. Even so, the 500 mm column experiment showed that there are a number of small impurities that can now be resolved from the main peaks. This is an opportunity to investigate these low abundant components further.

The volatile mobile phase selected is also mass spectrometry compatible and we suggest that our long column, small particle concept also applies for this detection technique.

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

3Thermo Scientific Poster Note • PN70153_E 06/12S

Table 2: Chromatography methods overview and peak capacity gains, black cohosh

Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products? Rainer Bauder1, Frank Steiner2, Michael Heidorn2, Markus Martin2, Fraser McLeod2 1Thermo Fisher Scientific, Chelmsford, MA; 2Thermo Fisher Scientific, Germering, Germany

Conclusion   Combining individual 150 mm or 250 mm UHPLC columns to achieve extended

column length and column volumes, is a feasible approach to boost separation of analytes found in complex samples such as botanical supplements.

  The approach is applicable to UV/VIS, charged aerosol, or mass spectrometry detection.

  Charged aerosol detection is a near universal responding detection technique to quantify components without chromophores and therefore an excellent addition to UV/VIS or MS detection.

  Improved resolution now enables the measurement of low abundant impurities that previously remained hidden by the more abundant analytes.

  Shallow gradients are able to provide improved separation for a wider range of samples and botanicals.

  The concept can also be applied to early drug discovery or drug development.

  Natural products from botanical sources can be better investigated and “fingerprinted” for adulteration testing and quality control. The concepts enable an in-depth assaying for relatively unknown samples and matrices.

References 1.  T. W. D. Chan; P. P. H. But; S. W. Cheng; I. M. Y. Kwok; F. W. Lau; and H. X. Xu;

"Differentiation and Authentication of Panax ginseng, Panax quin-quefolius, and Ginseng Products by Using HPCL/MS" Analytical Chemistry, 2000, 72 (10), 2329–2329.

2.  Avula B; Wang YH; Smillie TJ; Khan IA; "Quantitative determination of triterpenoids and formononetin in rhizomes of black cohosh (Actaea racemosa) and dietary supplements by using UPLC-UV/ELS detection and identification by UPLC-MS". Planta Med. 75 (4): 381–6. doi:10.1055/s-0028-1088384. PMID 19061153 (March 2009).

Overview Purpose: Evaluation of high peak capacity UHPLC setups and alternative, near universal detection technologies to improve separation and analysis of complex samples from natural sources.

Methods: Various serial arrangements of long, 2 µm particle UHPLC columns were used to investigate resolution improvements of selected botanical extracts.

Results: High efficiency UHPLC columns in 150 mm length (2 µm particles) already provide very high resolution chromatography. The use of longer UHPLC columns or the combination of 2 × 250 mm 2 µm C18 columns almost doubles peak capacities even for very complex samples. The improved resolution benefits component identification and isolation for further, in-depth investigation. This approach is a widely applicable strategy and is complemented by the near universal response of the Thermo Scientific Dionex Corona ultra RS detector.

Introduction

Natural product phytochemicals derived from botanical sources, are undergoing a revived interest in targeted drug development. In particular, traditional medicines from various geographies provide a truly global offering for promising leads. However, botanical samples often exhibit a significant complexity and many of their ingredients do not contain a chromophore or cannot readily be ionized. Hence, their analysis can be extremely challenging, both chromatographically and due to the limited ability to use UV absorbance and mass spectrometry detection.

High resolution UHPLC, using long columns packed with small particles and shallow gradients, is a very practical way to boost peak capacity. With the subsequent increase in resolution, small sized peaks eluting next to much larger ones can be more accurately integrated; thus characterization quality of complex mixtures is improved. This can be critical when a non-selective detector like the charged aerosol detector (Corona™ ultra RS™) is used. With the selection of herbal medicines taken from around the world, the effectiveness of using very long UHPLC columns with a wide range of detection techniques (charged aerosol detection, UV/VIS) is evaluated in this work.

Ginseng is any one of eleven species of slow-growing perennial plants with fleshy roots, belonging to the genus Panax of the family Araliaceae. Ginsenosides found at high abundance in the root are the purported active compounds that can be used to distinguish different Panax species. Herbal companies who follow Good Manufacturing Practices (GMP) regularly test for the quality, potency, and species authentication of their herbs. One published study found HPLC was especially useful in the differentiation and authentication of Panax ginseng from Panax quinquefolius due to the unambiguous distinction of slightly varying isotypes of ginsenoside compounds.1

Actaea racemosa (black cohosh, black bugbane, black snakeroot, fairy candle) is a plant from the buttercup family. Roots and rhizomes of black cohosh have long been used medicinally by Native Americans to treat gynecological and other disorders, including sore throats, kidney problems, and depression. Triterpenoids and formononetin in rhizomes of black cohosh have been previously studied and quantified using UHPLC and MS techniques.2

The milk thistle, silybum marianum, is a thistle of the genus Silybum Adans., a flowering plant of the daisy family (Asteraceae). They are native to the Mediterranean regions of Europe, North Africa and the Middle East. The name "milk thistle" derives from two features of the leaves; they are mottled with splashes of white and they contain a milky sap. Research is being undertaken on the physiological effects, potential therapeutic properties, and possible medical uses of milk thistle.

The above described, over-the-counter and readily available preparations have been selected to demonstrate the very practical and easy approach for boosting peak capacities and column efficiencies beyond commonly available UHPLC column formats. They also contain, if not proven but suggested, pharmaceutically active and valuable components which are likely to be studied further in the search of new active entities.

Methods

Sample Preparation

Herbal over-the-counter preparations of Korean white ginseng and black cohosh were extracted with methanol. About 500 mg of the capsules’ contents were sonicated for 15 min in 10 mL methanol (HPLC gradient grade). The milk thistle extract was prepared with the same approach using ca. 50 mg powdered content of the capsule in 5 mL methanol. The raw extracts were filtered through 0.45 µm syringe filters prior to transfer into the sample vials.

Liquid Chromatography System: Thermo Scientific Dionex UltiMate 3000 RSLC system

equipped with: LPG-3400XRS, DGP-3600RS, WPS-3000(X)RS, TCC-3000RS, DAD-3000RS, Corona ultra RS

Columns: Thermo Scientific Acclaim RSLC120 C18, 2.1mm ID, 2.2 µm particle, 150 mm, 250 mm and in combination

Mobile Phase A: 0.1% Formic acid in HPLC gradient grade water Mobile Phase B: 0.1% Formic acid in HPLC gradient grade methanol Flow Rate 0.40 mL/min Gradient Conditions: ginseng, cohosh => 45% B hold 3.0 min, 100% B in

20 min hold for 5.0 min, return and equilibrate for 7.0 min, other gradient times as indicated milk thistle => 17%B hold 3.0min, 43%B in 23.0min, 70% B in 0.1 min, hold for 5.0 min, 5.0 min to equilibrate

Injection Volume: 3–5 µL Corona ultra RS settings: Filter: 3 Nebulizer Temp On at 25 °C

Power Function Value 1.0

Mass Spectrometry

Mass spectrometry was not used for this evaluation.

Data Analysis

Thermo Scientific Dionex Chromeleon 7.1 SR.1 Chromatography Data System (CDS) was used for all data processing.

Results

Peak Capacity Improvements When Increasing Column Length While Maintaining Relative Gradient Time

Further improvement of resolution, even when having already applied 2 µm or sub 2 µm particle columns, is often only possible by adding column length. This approach has multiple benefits. First, the method does not need to be redeveloped as gradient times and slopes change relative to the added column length. For example, Table 1 shows, that the peak capacity for the ginseng extract jumps from 142 (40 °C, 150 mm) to 335 (75° C, 500 mm). Even when maintaining the original column temperature, the extension of the original 150 mm column length to 400 mm almost doubles peak capacity. Table 2 shows the results for black cohosh extract. Comparable efficiency improvements would require to reduce the particle size by half, which in this example would demand a 1µm particle column.

Second, with increased column volume and capacity, more sample and sample matrix are tolerated by the column. Low abundant peaks will be resolved better from prominent main peaks as can be seen in figures 2, 4, and 5. The time axis for the displayed traces are not to scale and allow a good, visual comparison. Please refer to tables 1 and 2 for gradient times and overall runtimes (150 mm and 500 mm column length for Figures 1-5).

Using peak capacity as the measure for separation performance of a given LC or UHPLC method is a very practical way to assess achievable resolution. The theoretical plate count for single peaks can only imperfectly describe the overall separation success. For this reason, the peak capacity was calculated by dividing the active gradient time with the averaged base peak width of all peaks found. To maintain comparative peak identification results, constant peak detection parameters were chosen and applied to all injections of the same extract.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO70153_E 06/12S

FIGURE 1: Overlay Korean white ginseng, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150 mm = 20 min

FIGURE 5: Zoomed overlay milk thistle extract, UV-detection at 288 nm tgradient 500mm = 90 min, tgradient 150mm = 23 min

Improved peak capacity and separation as observed for ginseng was also found for black cohosh. This is clearly illustrated in the zoomed overlays in Figures 2 and 4.

Although the use of a longer column required a simple adjustment in gradient time and column temperature, no additional method development was necessary. This straight- forward approach can consequently very easily be introduced as a standard operating procedure for new projects and (unknown) samples (matrices).

For mass spectrometry, the increasing peak width as shown in tables 1 and 2, have also very practical and beneficial impacts. While chromatographic resolution overall is improved, there is also more time to acquire data points. This improves the likelihood to measure and detect minor peaks. With fast collecting fraction collectors (2D-offline methods or 2D-comprehensive LC methods), trace and low abundant components can be further enriched and investigated.

Core enhanced particle UHPLC columns offer another way to improve peak sharpness and resolution. The chromatographer benefits from the faster mass transfer between mobile phase and stationary phase. Typically, this has to be traded off with reduced backpressure stability or reduced column loading capacity.

FIGURE 3: Overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

Table 1: Chromatography methods overview and peak capacity gains, Korean white ginseng

FIGURE 4: Zoomed overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

FIGURE 2: Zoomed overlay Korean white ginseng, Corona ultra RS tgradient 500mm = 66min, tgradient 150mm = 20min

In the case of the milk thistle extract, the sample was much less complex. Even so, the 500 mm column experiment showed that there are a number of small impurities that can now be resolved from the main peaks. This is an opportunity to investigate these low abundant components further.

The volatile mobile phase selected is also mass spectrometry compatible and we suggest that our long column, small particle concept also applies for this detection technique.

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

4 Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products?

Table 2: Chromatography methods overview and peak capacity gains, black cohosh

Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products? Rainer Bauder1, Frank Steiner2, Michael Heidorn2, Markus Martin2, Fraser McLeod2 1Thermo Fisher Scientific, Chelmsford, MA; 2Thermo Fisher Scientific, Germering, Germany

Conclusion   Combining individual 150 mm or 250 mm UHPLC columns to achieve extended

column length and column volumes, is a feasible approach to boost separation of analytes found in complex samples such as botanical supplements.

  The approach is applicable to UV/VIS, charged aerosol, or mass spectrometry detection.

  Charged aerosol detection is a near universal responding detection technique to quantify components without chromophores and therefore an excellent addition to UV/VIS or MS detection.

  Improved resolution now enables the measurement of low abundant impurities that previously remained hidden by the more abundant analytes.

  Shallow gradients are able to provide improved separation for a wider range of samples and botanicals.

  The concept can also be applied to early drug discovery or drug development.

  Natural products from botanical sources can be better investigated and “fingerprinted” for adulteration testing and quality control. The concepts enable an in-depth assaying for relatively unknown samples and matrices.

References 1.  T. W. D. Chan; P. P. H. But; S. W. Cheng; I. M. Y. Kwok; F. W. Lau; and H. X. Xu;

"Differentiation and Authentication of Panax ginseng, Panax quin-quefolius, and Ginseng Products by Using HPCL/MS" Analytical Chemistry, 2000, 72 (10), 2329–2329.

2.  Avula B; Wang YH; Smillie TJ; Khan IA; "Quantitative determination of triterpenoids and formononetin in rhizomes of black cohosh (Actaea racemosa) and dietary supplements by using UPLC-UV/ELS detection and identification by UPLC-MS". Planta Med. 75 (4): 381–6. doi:10.1055/s-0028-1088384. PMID 19061153 (March 2009).

Overview Purpose: Evaluation of high peak capacity UHPLC setups and alternative, near universal detection technologies to improve separation and analysis of complex samples from natural sources.

Methods: Various serial arrangements of long, 2 µm particle UHPLC columns were used to investigate resolution improvements of selected botanical extracts.

Results: High efficiency UHPLC columns in 150 mm length (2 µm particles) already provide very high resolution chromatography. The use of longer UHPLC columns or the combination of 2 × 250 mm 2 µm C18 columns almost doubles peak capacities even for very complex samples. The improved resolution benefits component identification and isolation for further, in-depth investigation. This approach is a widely applicable strategy and is complemented by the near universal response of the Thermo Scientific Dionex Corona ultra RS detector.

Introduction

Natural product phytochemicals derived from botanical sources, are undergoing a revived interest in targeted drug development. In particular, traditional medicines from various geographies provide a truly global offering for promising leads. However, botanical samples often exhibit a significant complexity and many of their ingredients do not contain a chromophore or cannot readily be ionized. Hence, their analysis can be extremely challenging, both chromatographically and due to the limited ability to use UV absorbance and mass spectrometry detection.

High resolution UHPLC, using long columns packed with small particles and shallow gradients, is a very practical way to boost peak capacity. With the subsequent increase in resolution, small sized peaks eluting next to much larger ones can be more accurately integrated; thus characterization quality of complex mixtures is improved. This can be critical when a non-selective detector like the charged aerosol detector (Corona™ ultra RS™) is used. With the selection of herbal medicines taken from around the world, the effectiveness of using very long UHPLC columns with a wide range of detection techniques (charged aerosol detection, UV/VIS) is evaluated in this work.

Ginseng is any one of eleven species of slow-growing perennial plants with fleshy roots, belonging to the genus Panax of the family Araliaceae. Ginsenosides found at high abundance in the root are the purported active compounds that can be used to distinguish different Panax species. Herbal companies who follow Good Manufacturing Practices (GMP) regularly test for the quality, potency, and species authentication of their herbs. One published study found HPLC was especially useful in the differentiation and authentication of Panax ginseng from Panax quinquefolius due to the unambiguous distinction of slightly varying isotypes of ginsenoside compounds.1

Actaea racemosa (black cohosh, black bugbane, black snakeroot, fairy candle) is a plant from the buttercup family. Roots and rhizomes of black cohosh have long been used medicinally by Native Americans to treat gynecological and other disorders, including sore throats, kidney problems, and depression. Triterpenoids and formononetin in rhizomes of black cohosh have been previously studied and quantified using UHPLC and MS techniques.2

The milk thistle, silybum marianum, is a thistle of the genus Silybum Adans., a flowering plant of the daisy family (Asteraceae). They are native to the Mediterranean regions of Europe, North Africa and the Middle East. The name "milk thistle" derives from two features of the leaves; they are mottled with splashes of white and they contain a milky sap. Research is being undertaken on the physiological effects, potential therapeutic properties, and possible medical uses of milk thistle.

The above described, over-the-counter and readily available preparations have been selected to demonstrate the very practical and easy approach for boosting peak capacities and column efficiencies beyond commonly available UHPLC column formats. They also contain, if not proven but suggested, pharmaceutically active and valuable components which are likely to be studied further in the search of new active entities.

Methods

Sample Preparation

Herbal over-the-counter preparations of Korean white ginseng and black cohosh were extracted with methanol. About 500 mg of the capsules’ contents were sonicated for 15 min in 10 mL methanol (HPLC gradient grade). The milk thistle extract was prepared with the same approach using ca. 50 mg powdered content of the capsule in 5 mL methanol. The raw extracts were filtered through 0.45 µm syringe filters prior to transfer into the sample vials.

Liquid Chromatography System: Thermo Scientific Dionex UltiMate 3000 RSLC system

equipped with: LPG-3400XRS, DGP-3600RS, WPS-3000(X)RS, TCC-3000RS, DAD-3000RS, Corona ultra RS

Columns: Thermo Scientific Acclaim RSLC120 C18, 2.1mm ID, 2.2 µm particle, 150 mm, 250 mm and in combination

Mobile Phase A: 0.1% Formic acid in HPLC gradient grade water Mobile Phase B: 0.1% Formic acid in HPLC gradient grade methanol Flow Rate 0.40 mL/min Gradient Conditions: ginseng, cohosh => 45% B hold 3.0 min, 100% B in

20 min hold for 5.0 min, return and equilibrate for 7.0 min, other gradient times as indicated milk thistle => 17%B hold 3.0min, 43%B in 23.0min, 70% B in 0.1 min, hold for 5.0 min, 5.0 min to equilibrate

Injection Volume: 3–5 µL Corona ultra RS settings: Filter: 3 Nebulizer Temp On at 25 °C

Power Function Value 1.0

Mass Spectrometry

Mass spectrometry was not used for this evaluation.

Data Analysis

Thermo Scientific Dionex Chromeleon 7.1 SR.1 Chromatography Data System (CDS) was used for all data processing.

Results

Peak Capacity Improvements When Increasing Column Length While Maintaining Relative Gradient Time

Further improvement of resolution, even when having already applied 2 µm or sub 2 µm particle columns, is often only possible by adding column length. This approach has multiple benefits. First, the method does not need to be redeveloped as gradient times and slopes change relative to the added column length. For example, Table 1 shows, that the peak capacity for the ginseng extract jumps from 142 (40 °C, 150 mm) to 335 (75° C, 500 mm). Even when maintaining the original column temperature, the extension of the original 150 mm column length to 400 mm almost doubles peak capacity. Table 2 shows the results for black cohosh extract. Comparable efficiency improvements would require to reduce the particle size by half, which in this example would demand a 1µm particle column.

Second, with increased column volume and capacity, more sample and sample matrix are tolerated by the column. Low abundant peaks will be resolved better from prominent main peaks as can be seen in figures 2, 4, and 5. The time axis for the displayed traces are not to scale and allow a good, visual comparison. Please refer to tables 1 and 2 for gradient times and overall runtimes (150 mm and 500 mm column length for Figures 1-5).

Using peak capacity as the measure for separation performance of a given LC or UHPLC method is a very practical way to assess achievable resolution. The theoretical plate count for single peaks can only imperfectly describe the overall separation success. For this reason, the peak capacity was calculated by dividing the active gradient time with the averaged base peak width of all peaks found. To maintain comparative peak identification results, constant peak detection parameters were chosen and applied to all injections of the same extract.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO70153_E 06/12S

FIGURE 1: Overlay Korean white ginseng, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150 mm = 20 min

FIGURE 5: Zoomed overlay milk thistle extract, UV-detection at 288 nm tgradient 500mm = 90 min, tgradient 150mm = 23 min

Improved peak capacity and separation as observed for ginseng was also found for black cohosh. This is clearly illustrated in the zoomed overlays in Figures 2 and 4.

Although the use of a longer column required a simple adjustment in gradient time and column temperature, no additional method development was necessary. This straight- forward approach can consequently very easily be introduced as a standard operating procedure for new projects and (unknown) samples (matrices).

For mass spectrometry, the increasing peak width as shown in tables 1 and 2, have also very practical and beneficial impacts. While chromatographic resolution overall is improved, there is also more time to acquire data points. This improves the likelihood to measure and detect minor peaks. With fast collecting fraction collectors (2D-offline methods or 2D-comprehensive LC methods), trace and low abundant components can be further enriched and investigated.

Core enhanced particle UHPLC columns offer another way to improve peak sharpness and resolution. The chromatographer benefits from the faster mass transfer between mobile phase and stationary phase. Typically, this has to be traded off with reduced backpressure stability or reduced column loading capacity.

FIGURE 3: Overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

Table 1: Chromatography methods overview and peak capacity gains, Korean white ginseng

FIGURE 4: Zoomed overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

FIGURE 2: Zoomed overlay Korean white ginseng, Corona ultra RS tgradient 500mm = 66min, tgradient 150mm = 20min

In the case of the milk thistle extract, the sample was much less complex. Even so, the 500 mm column experiment showed that there are a number of small impurities that can now be resolved from the main peaks. This is an opportunity to investigate these low abundant components further.

The volatile mobile phase selected is also mass spectrometry compatible and we suggest that our long column, small particle concept also applies for this detection technique.

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

5Thermo Scientific Poster Note • PN70153_E 06/12S

Table 2: Chromatography methods overview and peak capacity gains, black cohosh

Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products? Rainer Bauder1, Frank Steiner2, Michael Heidorn2, Markus Martin2, Fraser McLeod2 1Thermo Fisher Scientific, Chelmsford, MA; 2Thermo Fisher Scientific, Germering, Germany

Conclusion   Combining individual 150 mm or 250 mm UHPLC columns to achieve extended

column length and column volumes, is a feasible approach to boost separation of analytes found in complex samples such as botanical supplements.

  The approach is applicable to UV/VIS, charged aerosol, or mass spectrometry detection.

  Charged aerosol detection is a near universal responding detection technique to quantify components without chromophores and therefore an excellent addition to UV/VIS or MS detection.

  Improved resolution now enables the measurement of low abundant impurities that previously remained hidden by the more abundant analytes.

  Shallow gradients are able to provide improved separation for a wider range of samples and botanicals.

  The concept can also be applied to early drug discovery or drug development.

  Natural products from botanical sources can be better investigated and “fingerprinted” for adulteration testing and quality control. The concepts enable an in-depth assaying for relatively unknown samples and matrices.

References 1.  T. W. D. Chan; P. P. H. But; S. W. Cheng; I. M. Y. Kwok; F. W. Lau; and H. X. Xu;

"Differentiation and Authentication of Panax ginseng, Panax quin-quefolius, and Ginseng Products by Using HPCL/MS" Analytical Chemistry, 2000, 72 (10), 2329–2329.

2.  Avula B; Wang YH; Smillie TJ; Khan IA; "Quantitative determination of triterpenoids and formononetin in rhizomes of black cohosh (Actaea racemosa) and dietary supplements by using UPLC-UV/ELS detection and identification by UPLC-MS". Planta Med. 75 (4): 381–6. doi:10.1055/s-0028-1088384. PMID 19061153 (March 2009).

Overview Purpose: Evaluation of high peak capacity UHPLC setups and alternative, near universal detection technologies to improve separation and analysis of complex samples from natural sources.

Methods: Various serial arrangements of long, 2 µm particle UHPLC columns were used to investigate resolution improvements of selected botanical extracts.

Results: High efficiency UHPLC columns in 150 mm length (2 µm particles) already provide very high resolution chromatography. The use of longer UHPLC columns or the combination of 2 × 250 mm 2 µm C18 columns almost doubles peak capacities even for very complex samples. The improved resolution benefits component identification and isolation for further, in-depth investigation. This approach is a widely applicable strategy and is complemented by the near universal response of the Thermo Scientific Dionex Corona ultra RS detector.

Introduction

Natural product phytochemicals derived from botanical sources, are undergoing a revived interest in targeted drug development. In particular, traditional medicines from various geographies provide a truly global offering for promising leads. However, botanical samples often exhibit a significant complexity and many of their ingredients do not contain a chromophore or cannot readily be ionized. Hence, their analysis can be extremely challenging, both chromatographically and due to the limited ability to use UV absorbance and mass spectrometry detection.

High resolution UHPLC, using long columns packed with small particles and shallow gradients, is a very practical way to boost peak capacity. With the subsequent increase in resolution, small sized peaks eluting next to much larger ones can be more accurately integrated; thus characterization quality of complex mixtures is improved. This can be critical when a non-selective detector like the charged aerosol detector (Corona™ ultra RS™) is used. With the selection of herbal medicines taken from around the world, the effectiveness of using very long UHPLC columns with a wide range of detection techniques (charged aerosol detection, UV/VIS) is evaluated in this work.

Ginseng is any one of eleven species of slow-growing perennial plants with fleshy roots, belonging to the genus Panax of the family Araliaceae. Ginsenosides found at high abundance in the root are the purported active compounds that can be used to distinguish different Panax species. Herbal companies who follow Good Manufacturing Practices (GMP) regularly test for the quality, potency, and species authentication of their herbs. One published study found HPLC was especially useful in the differentiation and authentication of Panax ginseng from Panax quinquefolius due to the unambiguous distinction of slightly varying isotypes of ginsenoside compounds.1

Actaea racemosa (black cohosh, black bugbane, black snakeroot, fairy candle) is a plant from the buttercup family. Roots and rhizomes of black cohosh have long been used medicinally by Native Americans to treat gynecological and other disorders, including sore throats, kidney problems, and depression. Triterpenoids and formononetin in rhizomes of black cohosh have been previously studied and quantified using UHPLC and MS techniques.2

The milk thistle, silybum marianum, is a thistle of the genus Silybum Adans., a flowering plant of the daisy family (Asteraceae). They are native to the Mediterranean regions of Europe, North Africa and the Middle East. The name "milk thistle" derives from two features of the leaves; they are mottled with splashes of white and they contain a milky sap. Research is being undertaken on the physiological effects, potential therapeutic properties, and possible medical uses of milk thistle.

The above described, over-the-counter and readily available preparations have been selected to demonstrate the very practical and easy approach for boosting peak capacities and column efficiencies beyond commonly available UHPLC column formats. They also contain, if not proven but suggested, pharmaceutically active and valuable components which are likely to be studied further in the search of new active entities.

Methods

Sample Preparation

Herbal over-the-counter preparations of Korean white ginseng and black cohosh were extracted with methanol. About 500 mg of the capsules’ contents were sonicated for 15 min in 10 mL methanol (HPLC gradient grade). The milk thistle extract was prepared with the same approach using ca. 50 mg powdered content of the capsule in 5 mL methanol. The raw extracts were filtered through 0.45 µm syringe filters prior to transfer into the sample vials.

Liquid Chromatography System: Thermo Scientific Dionex UltiMate 3000 RSLC system

equipped with: LPG-3400XRS, DGP-3600RS, WPS-3000(X)RS, TCC-3000RS, DAD-3000RS, Corona ultra RS

Columns: Thermo Scientific Acclaim RSLC120 C18, 2.1mm ID, 2.2 µm particle, 150 mm, 250 mm and in combination

Mobile Phase A: 0.1% Formic acid in HPLC gradient grade water Mobile Phase B: 0.1% Formic acid in HPLC gradient grade methanol Flow Rate 0.40 mL/min Gradient Conditions: ginseng, cohosh => 45% B hold 3.0 min, 100% B in

20 min hold for 5.0 min, return and equilibrate for 7.0 min, other gradient times as indicated milk thistle => 17%B hold 3.0min, 43%B in 23.0min, 70% B in 0.1 min, hold for 5.0 min, 5.0 min to equilibrate

Injection Volume: 3–5 µL Corona ultra RS settings: Filter: 3 Nebulizer Temp On at 25 °C

Power Function Value 1.0

Mass Spectrometry

Mass spectrometry was not used for this evaluation.

Data Analysis

Thermo Scientific Dionex Chromeleon 7.1 SR.1 Chromatography Data System (CDS) was used for all data processing.

Results

Peak Capacity Improvements When Increasing Column Length While Maintaining Relative Gradient Time

Further improvement of resolution, even when having already applied 2 µm or sub 2 µm particle columns, is often only possible by adding column length. This approach has multiple benefits. First, the method does not need to be redeveloped as gradient times and slopes change relative to the added column length. For example, Table 1 shows, that the peak capacity for the ginseng extract jumps from 142 (40 °C, 150 mm) to 335 (75° C, 500 mm). Even when maintaining the original column temperature, the extension of the original 150 mm column length to 400 mm almost doubles peak capacity. Table 2 shows the results for black cohosh extract. Comparable efficiency improvements would require to reduce the particle size by half, which in this example would demand a 1µm particle column.

Second, with increased column volume and capacity, more sample and sample matrix are tolerated by the column. Low abundant peaks will be resolved better from prominent main peaks as can be seen in figures 2, 4, and 5. The time axis for the displayed traces are not to scale and allow a good, visual comparison. Please refer to tables 1 and 2 for gradient times and overall runtimes (150 mm and 500 mm column length for Figures 1-5).

Using peak capacity as the measure for separation performance of a given LC or UHPLC method is a very practical way to assess achievable resolution. The theoretical plate count for single peaks can only imperfectly describe the overall separation success. For this reason, the peak capacity was calculated by dividing the active gradient time with the averaged base peak width of all peaks found. To maintain comparative peak identification results, constant peak detection parameters were chosen and applied to all injections of the same extract.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO70153_E 06/12S

FIGURE 1: Overlay Korean white ginseng, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150 mm = 20 min

FIGURE 5: Zoomed overlay milk thistle extract, UV-detection at 288 nm tgradient 500mm = 90 min, tgradient 150mm = 23 min

Improved peak capacity and separation as observed for ginseng was also found for black cohosh. This is clearly illustrated in the zoomed overlays in Figures 2 and 4.

Although the use of a longer column required a simple adjustment in gradient time and column temperature, no additional method development was necessary. This straight- forward approach can consequently very easily be introduced as a standard operating procedure for new projects and (unknown) samples (matrices).

For mass spectrometry, the increasing peak width as shown in tables 1 and 2, have also very practical and beneficial impacts. While chromatographic resolution overall is improved, there is also more time to acquire data points. This improves the likelihood to measure and detect minor peaks. With fast collecting fraction collectors (2D-offline methods or 2D-comprehensive LC methods), trace and low abundant components can be further enriched and investigated.

Core enhanced particle UHPLC columns offer another way to improve peak sharpness and resolution. The chromatographer benefits from the faster mass transfer between mobile phase and stationary phase. Typically, this has to be traded off with reduced backpressure stability or reduced column loading capacity.

FIGURE 3: Overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

Table 1: Chromatography methods overview and peak capacity gains, Korean white ginseng

FIGURE 4: Zoomed overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

FIGURE 2: Zoomed overlay Korean white ginseng, Corona ultra RS tgradient 500mm = 66min, tgradient 150mm = 20min

In the case of the milk thistle extract, the sample was much less complex. Even so, the 500 mm column experiment showed that there are a number of small impurities that can now be resolved from the main peaks. This is an opportunity to investigate these low abundant components further.

The volatile mobile phase selected is also mass spectrometry compatible and we suggest that our long column, small particle concept also applies for this detection technique.

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

6 Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products?

Table 2: Chromatography methods overview and peak capacity gains, black cohosh

Can High Peak Capacity and Universal Detection Solve the Challenges in LC Characterization of Botanicals and Natural Products? Rainer Bauder1, Frank Steiner2, Michael Heidorn2, Markus Martin2, Fraser McLeod2 1Thermo Fisher Scientific, Chelmsford, MA; 2Thermo Fisher Scientific, Germering, Germany

Conclusion   Combining individual 150 mm or 250 mm UHPLC columns to achieve extended

column length and column volumes, is a feasible approach to boost separation of analytes found in complex samples such as botanical supplements.

  The approach is applicable to UV/VIS, charged aerosol, or mass spectrometry detection.

  Charged aerosol detection is a near universal responding detection technique to quantify components without chromophores and therefore an excellent addition to UV/VIS or MS detection.

  Improved resolution now enables the measurement of low abundant impurities that previously remained hidden by the more abundant analytes.

  Shallow gradients are able to provide improved separation for a wider range of samples and botanicals.

  The concept can also be applied to early drug discovery or drug development.

  Natural products from botanical sources can be better investigated and “fingerprinted” for adulteration testing and quality control. The concepts enable an in-depth assaying for relatively unknown samples and matrices.

References 1.  T. W. D. Chan; P. P. H. But; S. W. Cheng; I. M. Y. Kwok; F. W. Lau; and H. X. Xu;

"Differentiation and Authentication of Panax ginseng, Panax quin-quefolius, and Ginseng Products by Using HPCL/MS" Analytical Chemistry, 2000, 72 (10), 2329–2329.

2.  Avula B; Wang YH; Smillie TJ; Khan IA; "Quantitative determination of triterpenoids and formononetin in rhizomes of black cohosh (Actaea racemosa) and dietary supplements by using UPLC-UV/ELS detection and identification by UPLC-MS". Planta Med. 75 (4): 381–6. doi:10.1055/s-0028-1088384. PMID 19061153 (March 2009).

Overview Purpose: Evaluation of high peak capacity UHPLC setups and alternative, near universal detection technologies to improve separation and analysis of complex samples from natural sources.

Methods: Various serial arrangements of long, 2 µm particle UHPLC columns were used to investigate resolution improvements of selected botanical extracts.

Results: High efficiency UHPLC columns in 150 mm length (2 µm particles) already provide very high resolution chromatography. The use of longer UHPLC columns or the combination of 2 × 250 mm 2 µm C18 columns almost doubles peak capacities even for very complex samples. The improved resolution benefits component identification and isolation for further, in-depth investigation. This approach is a widely applicable strategy and is complemented by the near universal response of the Thermo Scientific Dionex Corona ultra RS detector.

Introduction

Natural product phytochemicals derived from botanical sources, are undergoing a revived interest in targeted drug development. In particular, traditional medicines from various geographies provide a truly global offering for promising leads. However, botanical samples often exhibit a significant complexity and many of their ingredients do not contain a chromophore or cannot readily be ionized. Hence, their analysis can be extremely challenging, both chromatographically and due to the limited ability to use UV absorbance and mass spectrometry detection.

High resolution UHPLC, using long columns packed with small particles and shallow gradients, is a very practical way to boost peak capacity. With the subsequent increase in resolution, small sized peaks eluting next to much larger ones can be more accurately integrated; thus characterization quality of complex mixtures is improved. This can be critical when a non-selective detector like the charged aerosol detector (Corona™ ultra RS™) is used. With the selection of herbal medicines taken from around the world, the effectiveness of using very long UHPLC columns with a wide range of detection techniques (charged aerosol detection, UV/VIS) is evaluated in this work.

Ginseng is any one of eleven species of slow-growing perennial plants with fleshy roots, belonging to the genus Panax of the family Araliaceae. Ginsenosides found at high abundance in the root are the purported active compounds that can be used to distinguish different Panax species. Herbal companies who follow Good Manufacturing Practices (GMP) regularly test for the quality, potency, and species authentication of their herbs. One published study found HPLC was especially useful in the differentiation and authentication of Panax ginseng from Panax quinquefolius due to the unambiguous distinction of slightly varying isotypes of ginsenoside compounds.1

Actaea racemosa (black cohosh, black bugbane, black snakeroot, fairy candle) is a plant from the buttercup family. Roots and rhizomes of black cohosh have long been used medicinally by Native Americans to treat gynecological and other disorders, including sore throats, kidney problems, and depression. Triterpenoids and formononetin in rhizomes of black cohosh have been previously studied and quantified using UHPLC and MS techniques.2

The milk thistle, silybum marianum, is a thistle of the genus Silybum Adans., a flowering plant of the daisy family (Asteraceae). They are native to the Mediterranean regions of Europe, North Africa and the Middle East. The name "milk thistle" derives from two features of the leaves; they are mottled with splashes of white and they contain a milky sap. Research is being undertaken on the physiological effects, potential therapeutic properties, and possible medical uses of milk thistle.

The above described, over-the-counter and readily available preparations have been selected to demonstrate the very practical and easy approach for boosting peak capacities and column efficiencies beyond commonly available UHPLC column formats. They also contain, if not proven but suggested, pharmaceutically active and valuable components which are likely to be studied further in the search of new active entities.

Methods

Sample Preparation

Herbal over-the-counter preparations of Korean white ginseng and black cohosh were extracted with methanol. About 500 mg of the capsules’ contents were sonicated for 15 min in 10 mL methanol (HPLC gradient grade). The milk thistle extract was prepared with the same approach using ca. 50 mg powdered content of the capsule in 5 mL methanol. The raw extracts were filtered through 0.45 µm syringe filters prior to transfer into the sample vials.

Liquid Chromatography System: Thermo Scientific Dionex UltiMate 3000 RSLC system

equipped with: LPG-3400XRS, DGP-3600RS, WPS-3000(X)RS, TCC-3000RS, DAD-3000RS, Corona ultra RS

Columns: Thermo Scientific Acclaim RSLC120 C18, 2.1mm ID, 2.2 µm particle, 150 mm, 250 mm and in combination

Mobile Phase A: 0.1% Formic acid in HPLC gradient grade water Mobile Phase B: 0.1% Formic acid in HPLC gradient grade methanol Flow Rate 0.40 mL/min Gradient Conditions: ginseng, cohosh => 45% B hold 3.0 min, 100% B in

20 min hold for 5.0 min, return and equilibrate for 7.0 min, other gradient times as indicated milk thistle => 17%B hold 3.0min, 43%B in 23.0min, 70% B in 0.1 min, hold for 5.0 min, 5.0 min to equilibrate

Injection Volume: 3–5 µL Corona ultra RS settings: Filter: 3 Nebulizer Temp On at 25 °C

Power Function Value 1.0

Mass Spectrometry

Mass spectrometry was not used for this evaluation.

Data Analysis

Thermo Scientific Dionex Chromeleon 7.1 SR.1 Chromatography Data System (CDS) was used for all data processing.

Results

Peak Capacity Improvements When Increasing Column Length While Maintaining Relative Gradient Time

Further improvement of resolution, even when having already applied 2 µm or sub 2 µm particle columns, is often only possible by adding column length. This approach has multiple benefits. First, the method does not need to be redeveloped as gradient times and slopes change relative to the added column length. For example, Table 1 shows, that the peak capacity for the ginseng extract jumps from 142 (40 °C, 150 mm) to 335 (75° C, 500 mm). Even when maintaining the original column temperature, the extension of the original 150 mm column length to 400 mm almost doubles peak capacity. Table 2 shows the results for black cohosh extract. Comparable efficiency improvements would require to reduce the particle size by half, which in this example would demand a 1µm particle column.

Second, with increased column volume and capacity, more sample and sample matrix are tolerated by the column. Low abundant peaks will be resolved better from prominent main peaks as can be seen in figures 2, 4, and 5. The time axis for the displayed traces are not to scale and allow a good, visual comparison. Please refer to tables 1 and 2 for gradient times and overall runtimes (150 mm and 500 mm column length for Figures 1-5).

Using peak capacity as the measure for separation performance of a given LC or UHPLC method is a very practical way to assess achievable resolution. The theoretical plate count for single peaks can only imperfectly describe the overall separation success. For this reason, the peak capacity was calculated by dividing the active gradient time with the averaged base peak width of all peaks found. To maintain comparative peak identification results, constant peak detection parameters were chosen and applied to all injections of the same extract.

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO70153_E 06/12S

FIGURE 1: Overlay Korean white ginseng, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150 mm = 20 min

FIGURE 5: Zoomed overlay milk thistle extract, UV-detection at 288 nm tgradient 500mm = 90 min, tgradient 150mm = 23 min

Improved peak capacity and separation as observed for ginseng was also found for black cohosh. This is clearly illustrated in the zoomed overlays in Figures 2 and 4.

Although the use of a longer column required a simple adjustment in gradient time and column temperature, no additional method development was necessary. This straight- forward approach can consequently very easily be introduced as a standard operating procedure for new projects and (unknown) samples (matrices).

For mass spectrometry, the increasing peak width as shown in tables 1 and 2, have also very practical and beneficial impacts. While chromatographic resolution overall is improved, there is also more time to acquire data points. This improves the likelihood to measure and detect minor peaks. With fast collecting fraction collectors (2D-offline methods or 2D-comprehensive LC methods), trace and low abundant components can be further enriched and investigated.

Core enhanced particle UHPLC columns offer another way to improve peak sharpness and resolution. The chromatographer benefits from the faster mass transfer between mobile phase and stationary phase. Typically, this has to be traded off with reduced backpressure stability or reduced column loading capacity.

FIGURE 3: Overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

Table 1: Chromatography methods overview and peak capacity gains, Korean white ginseng

FIGURE 4: Zoomed overlay of black cohosh, Corona ultra RS tgradient 500 mm = 66 min, tgradient 150mm = 20 min

FIGURE 2: Zoomed overlay Korean white ginseng, Corona ultra RS tgradient 500mm = 66min, tgradient 150mm = 20min

In the case of the milk thistle extract, the sample was much less complex. Even so, the 500 mm column experiment showed that there are a number of small impurities that can now be resolved from the main peaks. This is an opportunity to investigate these low abundant components further.

The volatile mobile phase selected is also mass spectrometry compatible and we suggest that our long column, small particle concept also applies for this detection technique.

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

500 mm

150 mm

PN70153_E 06/12S

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