The Selection of an HPLC Column October

Transcription

1 The Selection of an HPLC Column 07. ctober

2 Long-Term Collaboration + 30 Years of Experience

3 GL Sciences Inc. HPLC Selection of an HPLC Column 3

4 Contents. How to Select an HPLC Column How to select your first choice column. How to Select an HPLC Column For Highly Polar Analytes Your first choice column via Reversed Phase Mode Your first choice column via HILIC Mode 3. How to Select an HPLC Column 3 For High-Throughput Analysis, UHPLC Methods Your first choice low carbon loading column 4

5 5 Contents 4. How to Select an HPLC Column 4 For Separation of Basic Analytes & its Related Substances, Process Impurities Your first choice high carbon loading column 5. How to Select an HPLC Column 5 ther stationary phases that may improve separation

6 In this Seminar Generation Brand Chemistry Latest InertSustain Series Inertsil 4 Series 008 Inertsil 3 Series 994 Particle Size(s) Surface Area (m/g) Pore Size Carbon Load (%) Endcapping InertSustain AQ-C8 C8.9, 3, 5 μm A 3 ~0 InertSustainSwift C8 C8.9, 3, 5 μm 00 00A 9 ~0 InertSustain C8 C8, 3, 5 μm A 4 ~0 InertSustain C8 C8 3, 5 μm A 8 ~0 InertSustainSwift C8 C8.9, 3, 5 μm 00 00A 6 ~0 InertSustain Phenylhexyl Phenylhexyl, 3, 5 μm A 9 ~0 ph Range InertSustain Phenyl Phenyl 3, 5 μm A 0 None ~7.5 InertSustain NH NH 3, 5 μm A 7 None ~7.5 InertSustain Amide Amide 3, 5 μm A 5 None ~8.5 InertSustain Cyano Cyanopropyl 3, 5 μm A 8 ~7.5 Inertsil DS-HL C8 3, 5 μm A 3 ~7.5 Inertsil DS-4 C8, 3, 5 μm A ~7.5 Inertsil C8-4 C8, 3, 5 μm A 5 ~7.5 Inertsil Amide Amide 3, 5 μm A 8 None ~7.5 Inertsil DS-3 C8, 3, 4, 5 μm A 5 ~7.5 Inertsil DS-SP C8 3, 5 μm A 8.5 ~7.5 Inertsil Ph-3 Phenyl, 3, 5 μm A 9.5 None ~7.5 Inertsil HILIC Diol 3, 5 μm A 0 None ~7.5 6

7 Contents. How to Select an HPLC Column How to select your first choice column. How to Select an HPLC Column For Highly Polar Analytes Your first choice column via Reversed Phase Mode Your first choice column via HILIC Mode 3. How to Select an HPLC Column 3 For High-Throughput Analysis, UHPLC Methods Your first choice low carbon loading column 7

8 8 Survey Results provided from LCGC Magazine The remaining 5 to 0 % are developed by * C8, Phenyl, HILIC, Amino, Silica, Cyano etc. The majority of 80 to 85 % are developed by * C8

9 9 What is your first choice column? To start with, choose a column that has the highest possibility to deliver a satisfactory results for your application/method. = Choose a versatile column that would provide satisfactory results no matter what type of compound is included in your sample. Select an DS column as it has the ability to retain and separate various type of compounds.

10 0 What is your first choice column? DS Column: Suitable for compounds having various polarity Analysis of Water-soluble Vitamins (B), Hydrophilic Compound FLU 0 0 CH 3 N N NH N + S CH 3 CH CH Analytical Conditions Column: Temp. : 40 Inertsil DS-3 (5μm, 50 x 4.6 mm I.D.) Detection: FL Ex 375 nm Em 440 nm Injection Vol.:0μL (Fluorescently-derivatized by a Post- Column Method) Eluent: A) CH 3 B) Phosphate Buffer A/B = /9, v/v Flow Rate: 0.8 ml/min. Thiamine. Hydroxyethylthiamine (HET)

11 What is your first choice column? DS Column: Suitable for compounds having various polarity Analysis of Coenzyme Q0, Hydrophobic Compound mau CH 3 CH 3 CH 3 CH 3 9 CH 3 CH 3 Analytical Conditions Column : Inertsil DS-3 (3μm, 50 x 4.6 mm I.D.) Eluent : A) CH 3 B) C H 5 A / B = 65 / 35, v/v Flow Rate :.0 ml/min Temp. : 40 Detection : UV 75nm Injection Vol. : 5μL. Coenzyme Q

12 Important Points on when Selecting an HPLC Column Choosing an DS column from the market can be a very difficult process. To efficiently develop your methods, please keep in mind of the following. Peaks merging due to a tailing of peak Basic Compound Acidic Compound 3 Chelating Compound 4 Retentivity 5 Back Pressure 6 Lot-to-Lot Reproducibility

13 3 End-capping An DS column is packed with silica that are chemically bonded with DS (ctadecylsilyl, C8) groups into a stainless steel hardware. However, there are some residual of unreacted silanol groups. Basic compounds will interact adversely with these silanol groups, resulting in causing poor peak shapes. To eliminate/remove these silanol groups, we employ a procedure called End-capping. C8 C8 C8 C8 C8 C8 Si Si Si Silica Gel Si Si Si Si Silica Gel Si C Si C Si Si Silica Gel C Si C Bonding of DS groups End-capping

14 Analysis of Basic Compounds When the end-capping is insufficient and the packing material contains residual silanol groups, basic compounds will interact adversely with those silanol groups. Columns that are treated with fine end-capping can only produce satisfactory peak shape for Dextromethorphan. InertSustain C Analytical Conditions Column :. mm I.D. 50 mm, 3 μm Eluent : CH 3 CN / 5mM Phosphate Buffer(pH=7.0) = 30 / 70 Col. Temp. : 40 Ditection : UV30 nm Flow Rate : 0. ml/min Sample : :Uracil :Pyridine 3:Phenol 4:Berberine chloride 5:Dextromethorpan N H 3 C CH 3 H N CH3 CH 3 Berberine chloride Dextromethorphan 4

15 Analysis of Basic Compounds L-column DS Luna 3u C8() X SELECT CSH C CAPCELL PAK C8 MGⅢ 5

16 Intensity, cps 6 Analysis of Basic Compounds Simultaneous Analysis of Antidepressants 9.00e5 8.00e5 7.00e5 6.00e5 5.00e5 4.00e5 3.00e5.00e5.00e5 3. Amitriptyline Analytical Conditions System : LC800 HPLC system 4000 Q TRAP Column : InertSustain C8 (3μm, 50 x. mm I.D.) Eluent : A) 0. % HC in CH 3 CN : B) 0. % HC in H : A/B = / min- 40/ min- 40/60,v/v Flow Rate : 0.4 ml/min Col. Temp. : 40 Detection : LC/MS/MS (4000Q TRAP : ESI, Positive, MRM) CUR CAD IS TEM GS GS Injection Vol. : μl Sample : Antidepressants (each 00 ng/ml) Sample:. Sulpilide (34/) 9. Nortriptyline (64/33). Milnacipran (47/30) 0. Maprotiline (78/50) 3. Trazodone (37/76).Amitriptyline (78/33) 4. Amoxapine (34/7). Trimmipramine (95/00) 5. Doxepin (80/07) 3. Fluoxetine (30/44) 6. Desipramine(67/7) 4. Sertraline (306/75) 7. Fluvoxamine(39/7) 5. Clomipramine (35/86) 8. Imipramine(8/86) Time, min

17 7 Analysis of Acidic Compounds Some DS columns in the market produces poor peak shapes for acidic compounds. This is due to the fact that there are some basic compounds available on the surface of the silica gel. Some columns introduces a basic group for their end-capping treatment to prevent the interaction between basic compounds. Tailing would be observed on Brilliant Blue FCF and Salicylic Acid when the surface of the packing material is basic. InertSustain C8 3 Analytical Conditions Column :. mm I.D. 50 mm, 3 μm Eluent : A) CH 3 CN B) 0.%H 3 P 4 A/B = 5/75, v/v Col. Temp. : 40 Detection : UV54 nm Flow Rate : 0. ml/min Sample :. Brilliant Blue FCF. Phenol 3. Salicylic acid S S 3 - S 3 - Brilliant Blue FCF N N +

18 Analysis of Acidic Compounds L-column DS Luna 3u C8() No peak 3 No peak X SELECT CSH C CAPCELL PAK C8 MGⅢ 8

19 9 Analysis of Chelating Compounds Hinokitiol is a strong chelating compound, which coordinately binds with the surface of residual trace impurities, resulting in severe tailing. InertSustain C8 TF= min st injection Analytical Conditions System : GL7400 Column : 5 μm mm I. D. Eluent : CH 3 CN / 0.% H 3 P 4 = 40 / 60 Flow rate :.0mL /min Col. Temp. :40 Detection :54 nm Sample :.Hinokitiol TF=. 8.33min nd injection

20 0 Analysis of Chelating Compounds The peak shape may improve as the injection increases since the surface of the packing material of the adsorption active sites eventually become masked. However, in this case, stable quantitative chromatograms cannot be expected. st injection nd injection X SELECT CSH C8

21 Cost From Column Manufacturer A Column Manufacture A For the Analysis of Basic Compounds Column Y USD $ Column Z USD $ For the Analysis of Acidic Compounds Total Cost USD $,000.00

22 Cost From GL Sciences GL Sciences InertSustain C8 USD $ Total Cost USD $ For the Analysis of Basic, Acidic and Chelating Compounds

23 3 High Quality at Reasonable Prices by 35 Year s of Experience Execution of whole manufacturing process with reliable and consistent high technologies is essential for the reproducibility of high column efficiency resulting in best customer satisfaction. This is also the only way to reduce the cost which enable us to supply our customer with HPLC columns at a reasonable price. Synthesis of Base Silica Gel from Pure Ingredients Execution of Whole Manufacturing Process Bonding of Functional Groups on the Silica Gel Media Packing of Silica Gel Media into HPLC Column Hardwares

24 4 Reliable Reproducibility & Quality Control Program Needless to mention not only the retentivity is controlled, but all other physical properties or characteristics are rigorously inspected as well to maintain consistent performance from column-to-column & lot-to-lot. Lot# A Lot# B Selectivity Test Analytical Conditions Column : InertSustain C8 4.6mmI.D. 50mm, 5μm Eluent : CH 3 / H = 80 / 0, v/v Flow Rate :.0 ml / min Col. Temp.: 40 Detection : UV 54 nm Sample : : Uracil : Caffeine 3: Phenol 4: Butylbenzene 5: o-terphenyl 6: Amylbenzene 7: Triphenylene Lot# C

25 5 Reliable Reproducibility & Quality Control Program Needless to mention not only the retentivity is controlled, but all other physical properties or characteristics are rigorously inspected as well to maintain consistent performance from column-to-column & lot-to-lot. Lot# A Inspection Test using Dextromethorphan TF=.0 Analytical Conditions Column :InertSustain C8 4.6 mm I.D. 50 mm, 5 μm Eluent :CH 3 CN / 5 mm Phosphate buffer(ph7.0) = 40 / 60 Col. Temp. :40 Ditection :UV 0 nm Flow Rate :.0 ml / min Sample : : Dextromethorpan Lot# B Lot# C TF=.8 TF=.7

26 6 Retentivity of an DS Column If the Retentivity is strong Ability to retain compounds using high concentration organic solvents It can offer variety of benefits for various applications

27 Difference of Retentivity depending on the Column Carbon Load Inertsil DS-3 Carbon Load: 5 % Analytical Conditions Column : 5μm, mm I.D. Eluent : CH 3 / H =80 / 0 Flow Rate :.0 ml/min Col.Temp. : 40 Detection : UV 54 nm InertSustain C8 Carbon Load: 4 % Inertsil DS ) Uracil ) Caffein 3) Phenol 4) n-butylbenzene 5) o-terphenyl 6) n-amylbenzene 7) Triphenylene Carbon Load: % Inertsil DS-SP Carbon Load: 8.5 % 0 0 ther Brands Column A

28 Relative Ratio of Intensity 8 Retentivity and Sensitivity in LC/MS Higher the Concentration of rganic Solvent, Higher the Sensitivity in LC/MS 4E+04 Meアミオダロン Amiodarone Intensity, cps 3E+04 3E+04 E+04 E+04 Meピンドロール Pindolol MeCN アミオダロン Amiodarone MeCN ピンドロール Pindolol HN I I Amiodarone N E+04 NH 5E+03 0E Concentration 有機溶媒濃度 of rganic (v/v) Solvent (v/v) Pindolol Evaluated by Infusion Method API-4000Q : MRM (ESI Posi)

29 容量 (ml) Volume (ml) Easy Removal of Solvents for Preparative HPLC Higher the Concentration of rganic Solvent, Easier to Remove Solvents % 0% 40% 60% 80% 00% Concentration Me 濃度 of Me Took 5mL of solvent and depressurized for 5 minutes and confirmed how much solvent was left 9

30 30 Physical Properties of InertSustain C8 Silica: Newly Developed Silica Gel Bonded Phase: ctadecyl Groups Bonded-Phase Structure: Monomeric Particle Size:, 3, 5 μm Surface Area: 350 m /g Pore Size: 00A (0 nm) Pore Volume: 0.85 ml/g Carbon Loading: 4 % End-capping: Complete ph Range: ~0 USP Code: L

31 3 How will InertSutain C8 help with your Critical Samples? Superior Peak Shape with Better Resolution As the adsorption sites and silanol distribution are strictly controlled on the new silica, InertSustain C8 delivers highly stable chromatograms for qualitative and quantitative analysis for all compounds. InertSustain C8 ther Brands C8 Wide ph Compatibility and High Durability Highly durable to acidic and basic mobile phases and can endure from ph to 0. Unmatched perating Back Pressure Delivers low pressure even using high-viscosity solvents. Strict Quality Control Program results in High Reproducibility Since the entire manufacturing process is under the control of our factory in Japan, reliable and consistent performance from column to column and lot to lot can be stably supplied.

32 3 DS Columns and Back Pressure When the Column Back Pressure is High Possibility to damage the HPLC instrument and column Resulting in damage or deterioration

33 33 What makes the column back pressure so different? It depends on the Size of the silica gel Uniformity of the particle size Particle shape 5μm Uniformed Spherical Back pressure = Low 3μm Uniformed Spherical Back pressure = High 5μm Not uniformed Spherical Back pressure=high 5μm Uniformed Irregular Back pressure=high

34 What makes the column back pressure so different? Packing -Low back pressure -High theoretical plates -Lot-to-Lot reproducibility InertSustain or Inertsil DS-3 Series Packing -High back pressure -Low theoretical plates -Poor Lot-to-Lot reproducibility Competitor Y s silica gel 34

35 35 Why is the particle size distribution different? Every commercial columns have a particle size distribution. But it s distribution width are quite different Commercial silica-gel InertSustain, Inertsil DS-3 Series Percentage(%) Percentage(%) Particle Size (μ m) Particle Size (μ m) Most column manufactures do not synthesize their silica-gel. They purchase commercial silica-gel from silica-gel specialty manufacture. GL Sciences synthesizes the original silica-gel focused for HPLC columns. As a result, the particle size distribution width is highly controlled.

36 36 Back Pressure of Various DS Columns in the Market Extremely Low perating Back Pressure

37 37 Back Pressure of Various DS Columns in the Market Confirmation of Lowest Back Pressure

38 38 What if the perating Back Pressure is High? Increase in running-cost. (Frequently replacing instrument spare parts/accessories) Force to Analyze at Low Flow Rate (Decrease in Efficiency, Less NTP)

39 39 Plus Something Extra: Durability of InertSustain C8 ffers wide ph compatibility and high durability. Some samples require high ph for dissolution or to maintain stability. InertSustain C8 would be the first choice for such applications. Purging Conditions Column :4.6 mm I.D. 50 mm, 5 μm Eluent :50 mm Triethylamine (ph0.0) / CH 3 = 70/30, v/v Flow Rate :.0 ml/min Col. Temp. :50 Analytical Conditions Eluent :CH 3 CN/H = 65/35, v/v Flow Rate :.0 ml/min Col. Temp. :40 Detection :UV54 nm Sample :Naphthalene

40 Durability to Alkaline Sample Solutions Due to the increase in candidate drug compounds & addition of legally controlled compounds, there is a demand on changing the ph of dissolving solvent to improve the stability and solubility of the sample. We therefore have conducted a durability test to alkaline sample solutions as follows. Testing Procedure: Naphthalene test was conducted after injecting 00 ul of alkaline solution (0. N Na, ph 3). The mobile phase is Water 00% and the column temperature at 50 C, when injecting the alkaline solution. Naphthalene test Injecting alkaline solution 5 times Naphthalene test Injecting alkaline solution 5 times Naphthalene test Injecting alkaline solution 0 times Naphthalene test Injecting alkaline solution 30 times Naphthalene test From this point, Naphthalene test was conducted after injecting an alkaline solution 50 times. Number of injections of alkaline sample solution Naphthalene Test Column : InertSustain C8 (5 μm, mm I.D.) Eluent : A) CH 3 CN : B) H A/B = 65/35, v/v (premix) Flow Rate : ml/min Col. Temp. : 40 Detection : UV 54 nm Injection Vol. : 5 μl Sample : Naphthalene 40

41 4 Inertness, the Most Important Factor in the Separation Analysis Perfect as a first choice column for your method development Why??? When the chemical structure of an eluted peak is unknown Unknown Peak Target Peak Unknown Peak Target Peak If the column is inert No matter the analytes are acidic, basic or chelating compounds Exceptional peak shapes can be obtained on wide range of compounds. Therefore, we strongly recommend to choose a highly inert column.

42 4 Summary of Selecting an HPLC Column Your first choice column shall be a versatile column, which is an DS column. Although there are many DS columns in the market, their selectivity or characteristics are different. Before purchasing a column, make sure to check and confirm its inertness, operating back pressure and retentivity.

43 Contents. How to Select an HPLC Column How to select your first choice column. How to Select an HPLC Column For Highly Polar Analytes Your first choice column via Reversed Phase Mode Your first choice column via HILIC Mode 3. How to Select an HPLC Column 3 For High-Throughput Analysis, UHPLC Methods Your first choice low carbon loading column 43

44 44 Method Development for Polar Compounds First Choice Column Conventional DS Column In case there were no retention of polar compounds. Use of ion-pairing reagents. Use of 00% water mobile phase ften experience shorter column lifetime, poor method reproducibility, increased instrument maintenance What other options are left?

45 45 Method Development for Polar Compounds Second Choice Columns 3. Use of different reversed phase columns Selecting different DS columns having different carbon loading % Selecting polar embedded DS columns Selecting different stationary phase columns such as Phenyl or PFP columns Really hard to tell if the polar analytes will be retained or not. Adsorbed peaks of target analytes occur due to secondary interaction.

46 46 Method Development for Polar Compounds 4. Use of different interactions Second Choice Columns Selecting mix mode columns (ion-exchange + DS) Selecting HILIC columns (hydrophilic interaction) Use of high concentration buffer (harsh mobile phase). Reproducibility issues. Takes time to equilibrate columns prior to the analysis.

47 47 Physical Properties InertSustain AQ-C8 Silica : ES Silica Gel Carbon Load : 3.0 % Particle Size :.9 μm, 3 μm, 5 μm End-capping : Yes Surface Area : 350 m /g ph Range : 0 Pore Size : 00 Å (0 nm) USP Code : L, L96 Pore Volume : 0.85 ml/g Bonded Phase : ctadecyl Groups Bonded-Phase Structure : * Monomeric * Although it is classified as a monomeric type DS, the bonding of DS groups are optimized to retain highly polar compounds.

48 Benefits of InertSustain AQ-C8 Exceptional polar compound retention in reversed phase mode The usage of highly inert packing with superior column lifetime True polar compound retention without using any secondary interaction Improve separation between polar analytes and solvent peaks or sample matrices Enhanced retention can also prevent ion suppression in LC/MS methods 48

49 49 Method Development for Polar Compounds How will InertSustain AQ-C8 columns help with your polar samples? Easier to develop and optimize analytical conditions ffer strong polar compound retention even under 00% water or water rich mobile phases 49

50 50 Method Development for Polar Compounds The usage of highly inert packing results in delivering symmetric peaks for virtually any type of compounds How will InertSustain AQ-C8 columns help with your polar samples? Eliminating the use of ion-pairing reagents improves method reproducibility, extends column lifetime and reduces instrument maintenance Same selectivity from.9 to 5 μm columns for compatibility with any LC 50

51 5 Comparison of Retention for Nucleosides Nucleosides are often analyzed in HILIC mode as they are highly hydrophilic. However, DS columns also show some retention under 00% water mobile phase. In the following test, InertSustain AQ-C8 demonstrated to show simply stronger retention without the change of selectivity and analytical condition. InertSustain AQ-C8 Atlantis T3 t t Conditions Column : 5 μm, mm I.D. Eluent : 0.% HC in H Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 54 nm Sample. Cytidine. Uridine 3. Adenosine 4. Guanosine 5. 5-Methyl uridine InertSustain C8 (Conventional DS) t

52 5 Comparison of Retention for Catechins (Neutral) As shown below, InertSustain AQ-C8 delivered stronger retention of catechins even under 0% organic solvent mobile phase with exceptional peak shapes, while competitive column brand failed. Furthermore, InertSustain AQ-C8 can prevent the co-elution between the targeted polar analytes and solvent peaks or sample matrices due to its enhanced retentivity. InertSustain AQ-C8 Atlantis T3 InertSustain C8 (Convetnional DS) Complete separation between solvent peak Elution with solvent peak Baseline separation 34 Unresolved peaks Conditions Column : 5 μm, mm I.D. Eluent : A) ACN B) 0.% HC in H A/B = 0 /80 Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 80 nm Injection Vol. : μl Sample:. Gallocatechin(-) (GC(-)). Catechin(+) (C) 3. Epicatechin (-) (EC) 4. Epigallocatechin gallate(-) (EGCg) 5. Epicatechin gallate(-) (ECg) 6. Catechin gallate(-) (Cg) 00 μg/ml each

53 53 Comparison of Retention for Coffee Samples (Acidic, Basic) InertSustain AQ-C8 demonstrated stronger retention for hydrophilic coffee samples (acidic, basic) under 0% organic solvent mobile phase. InertSustain AQ-C8 t0 3 Conditions Column Eluent : 5 μm, mm I.D. : A) ACN B) 0.% HC in H Atlantis T t0 3 A/B = 0 /80 Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 70 nm Injection Vol. : μl InertSustain C8 (Convetnional DS) t0 3 Sample. Chlorogenic acid 00 μg/ml (Acidic). Caffeine 00 μg/ml (Basic) 3. Caffeic acid 00 μg/ml (Acidic)

54 54 Comparison of Peak Shapes for Basic Compounds Dextromethorphan is a strong basic compound. Severe tailing can be confirmed when the packing material contains residual silanol groups. InertSustain AQ-C8 Atlantis T3 CAPCELL PAK C8-AQ Synergi Fusion RP Aquasil C Not eluted, 3 Not eluted, 3 Not eluted Conditions Eluent : A) CH 3 CN B) 5 mm K HP 4 (ph 7.0) A/B = 30/70,v/v Flow Rate : ml/min Col. Temp. : 40 Detection : UV 30 nm Sample :. Uracil. Berberine chloride 3. Dextromethorphan H 3 C N CH 3 H CH 3 N CH3. Berberine chloride 3. Dextromethorphan

55 55 Comparison of Peak Shapes for Acidic Compounds Sharp peaks can be obtained when analyzing Phenol or Salicylic Acid. However, as Brilliant Blue FCF has three sulfonic groups in its chemical structure, tailing will occur when the surface of the packing material is slightly basic. InertSustain AQ-C8 Atlantis T3 CAPCELL PAK C8-AQ Synergi Fusion RP Aquasil C8 Not eluted Not eluted Not eluted Conditions Eluent : A) CH 3 CN B) 0.% H 3 P 4 in H A/B = 5/75,v/v Flow Rate : ml/min Col. Temp. : 40 Detection Sample : UV 54 nm :. Brilliant Blue FCF. Phenol 3. Salicylic acid - 3 S N - S 3 N +. Brilliant Blue FCF S 3 -

56 56 Comparison of Peak Shapes for Chelating Compounds Hinokitiol and Piroctone lamine are strong chelating compounds, which coordinately binds with the surface of residual trace metal impurities, resulting in severe tailing. However, the peak shape improves as the injection increases since the surface of the packing material of the adsorption active sites eventually become masked. InertSustain AQ-C8 Atlantis T3 CAPCELL PAK C8-AQ Synergi Fusion RP Aquasil C8 : Split peak : Split peak 3 3 Not eluted 3 Not eluted, 3 Not eluted Conditions Eluent : A) CH 3 CN B) 0.%HC in H A/B = 40/60 Flow Rate : ml/min Col. Temp. : 40 Detection : UV 30 nm Sample :. 8-Quinolinol. Hinokitiol 3. Piroctone lamine N. 8-Quinolino. Hinokitiol 3. Piroctone lamine N + H NH

57 Comparison of Compatibility with 00% Water Mobile Phase When analyzing hydrophilic compounds under water rich mobile phase condition, once the pump is stopped, the hydrophobic bonded group pushes the aqueous mobile phase out off the pore in an irreversible fashion, in what has become known as the dewetting phenomenon. InertSustain AQ-C8 guarantees compatibility with 00% aqueous mobile phases without the risk of dewetting. Testing Procedure: ) 00 % water is introduced into column over 60 minutes. ) Conduct Analysis ( st injection). 3) Stop flow for 5 minutes. 4) 00 % water is introduced again into column over 30 minutes. 5) Conduct Analysis ( nd injection). 6) Stop flow for 5 minutes again. 7) 00 % water is introduced again into column over 30 minutes. 8) Conduct Analysis (3 rd injection). 9) Disconnect the column from the system and stored for 4 hrs. 0) 00 % water is introduced into column over 30 minutes. ) Conduct Analysis (4 th injection). Conditions Eluent : H (00%) Flow Rate : ml/min Col. Temp. : 40 Detection : UV 54 nm Sample :. Cytosine. Uracil 3. Guanine 4. Thymine 5. Adenine 57

58 58 What is Dewetting? 00% aqueous mobile phase Pore With flow and pressure 3 Stopping the flow, with no pressure Restart flow Pores dewet. - Analytes never enter the pore. - Resulting in no retention. hydrophobic bonded phase Flush with rganic Solvent to Rewet Ex: Methanol

59 Comparison of Compatibility with 00% Water Mobile Phase InertSustain AQ-C8 Atlantis T3 st Injection Testing Procedure: ) 00 % water is introduced into column over 60 minutes. ) Conduct Analysis ( st injection). 3) Stop flow for 5 minutes. 4) 00 % water is introduced again into column over 30 minutes. 5) Conduct Analysis ( nd injection). 6) Stop flow for 5 minutes again. 7) 00 % water is introduced again into column over 30 minutes. 8) Conduct Analysis (3 rd injection). 9) Disconnect the column from the system and stored for 4 hrs. 0) 00 % water is introduced into column over 30 minutes. ) Conduct Analysis (4 th injection). 3 rd Injection.3% Retention Lost % Retention Lost Conditions Eluent : H (00%) Flow Rate : ml/min Col. Temp. : 40 Detection : UV 54 nm 4 th Injection.3% Retention Lost 6.% Retention Lost Sample :. Cytosine. Uracil 3. Guanine 4. Thymine 5. Adenine

60 60 Extended Column Lifetime at Low ph Durability Test at Low ph (ph.0) 理論段数 Efficiency (N) 保持時間 Retention Time Purging Conditions: Column :5 μm, mm I.D. Eluent : CH 3 CN/ H / TFA (0/90/), v/v/v (ph ) Column Temp. : 60 Analytical Conditions: Eluent Flow Rate Col. Temp. :40 Detection Sample :CH 3 CN/H = (65/35), v/v :.0 ml / min :UV 54 nm :Naphthalene Results: Retention Time Efficiency (N) :nly 0.7% Retention Lost :nly 0.% Efficiency Lost

61 6 Extended Column Lifetime at High ph Durability Test at High ph (ph 9.5) Purging Conditions: Column :5 μm, mm I.D. Eluent :50 mm TEA (ph 9.5)/CH 3 = 70/30, v/v Flow Rate :.0 ml/min Column Temp. : 理論段数維持率 Efficiency (N) 保持時間 Retention Time Analytical Conditions: Eluent Flow Rate Col. Temp. :40 Detection Sample :CH 3 CN/H = (65/35), v/v :.0 ml / min :UV 54 nm :Naphthalene Results: Retention Time Efficiency (N) :nly 5.8% Retention Lost :nly 0.6% Efficiency Lost

62 6 Extended Column Lifetime under 00 % Water Mobile Phase st Injection Injections Conditions Column : 5 μm, 4.6 mm I.D x 50 mm Eluent : 50mM CH 3 CNH 4 in H (ph 6.0) Flow Rate :.0 ml / min Col. Temp.: 60 Detection : UV 54 nm Sample. Cytosine. Uracil 3. Guanine 4. Thymine 5. Adenine

63 63 Extended Column Lifetime under 00 % Water Mobile Phase Conditions Column : 5 μm, 4.6 mm I.D 50 mm Eluent : 50mM CH 3 CNH 4 in H (ph 6.0) Flow Rate :.0 ml / min Col. Temp.: 60 Detection : UV 54 nm Sample :. Cytosine. Uracil 3. Guanine 4. Thymine 5. Adenine

64 64 Simple Method Transfer from HPLC to UHPLC 5 μm μm mm mm Conditions Column :5 μm, mm I.D. or.9 μm, 50. mm I.D. Eluent: A: 0.% H 3 P 4 B: CH 3 CN 5 μm: A/B = 99/ (5 min)- 99/ (5min) 80/0 (5min hold) -(min) - /99 (40 min) (min) - 99/.9 μm: A/B = 99/ ( min)- 80/0 (. min hold)- (0. min)- 99/ Flow Rate :.0 ml/min (5um) or 0.4 ml/min (.9μm) Col. Temp. :40 Detection :UV 0 nm Sample :. Pyridoxamine. Thiamin 3. Nicotinic Acid 4. Ascorbic acid 5. Nicotinamide 6. Pyridoxal 7. Pyridoxine 8. Pantothenic acid 9. Folic Acid 0. cyanocobalamin. Riboflavin. Biotin

65 65 Simple Method Transfer from HPLC to UHPLC Selectivity Test Retention of Polar Analytes (Nucleoside).9 μm. 50 mm μm. 50 mm μm. 50 mm Conditions Eluent : CH 3 /H = 80/0, v/v Flow Rate : 0.4 ml/min Col. Temp. : 40 Detection : UV 54 nm Sample : : Uracil :Cafeine 3. Phenol 4: Butylbenzen 5. o-terphenyl 6. Amylbenzene 7. Triphenylene Conditions Eluent : 0.% HC in H Flow Rate : 0.4 ml/min Col. Temp. : 40 Detection : UV 54 nm Sample :. Cytidine. Uridine 3. Adenosine 4. Guanosine 5. 5-Methyluridne

66 66 Comparison of Peak Shapes for Basic Compounds using Sub- um Columns InertSustian AQ-C8 (.9 μm) 3 N:935 k: 5.8 N3:800 k: ACQUITY UPLC HSS T3 (.8 μm) N:4339 k:5.3 3 N3:33 k: ACQUITY UPLC BEH C8 (.7 μm) N:867 k:.7 N3:5764 k: Conditions Eluent : A) CH 3 CN B) 5 mm K HP 4 (ph 7.0, KH P 4 ) A/B = 30/70,v/v Flow Rate : 0.4 ml/min Col. Temp. : 40 Detection : UV 30 nm Sample :. Uracil. Berberine chloride 3. Dextromethorphan Conditions Eluent : A) CH 3 CN B) 5 mm K HP 4 (ph 7.0, KH P 4 ) A/B = 60/40,v/v Flow Rate : 0.4mL/min Col. Temp. : 40 Detection : UV 0 nm Sample :. Chlorpheniramine. Triprolidine 3. Homochlorcyclizine 4. Hydroxyzine 5. Clemastine

67 67 Summary Your First Choice Column for For Highly Polar Analytes InertSustain AQ-C8 *Easier to develop and optimize analytical conditions *ffer strong polar compound retention even under 00% water or water rich mobile phases *The usage of highly inert packing results in delivering symmetric peaks for virtually any type of compounds *Eliminating the use of ion-pairing reagents improves method reproducibility, extends column lifetime and reduces instrument maintenance

68 68 HILIC Mode What if the sample could not be retained or require more retention of sample under reversed phase mode? Consider changing the separation mode to HILIC using HILIC columns

69 When to Select HILIC Mode How to select the Separation Mode Hydrophilic Polarity of Target Compound Hydrophobic When the sample is highly polar and cannot be retained using a reversed phase mode. HILIC Ion-Pair Reagent + Reversed Phase Reversed Phase Partition Chromatography Normal Phase Mainly used Solvents Acetonitrile Water Mainly used Solvents Acetonitrile Methanol THF Water When the sample contains similar hydrophobicity and Cannot be retained using a reversed phase mode. Mainly used Solvents n-hexane Ethyl Acetate IPA Ethanol 69

70 70 HILIC Mode Usage of HILIC mode with InertSustain Amide HILIC (Hydrophilic Interaction Chromatography) Benefits of HILIC mode The stationary phase is highly polar Excellent for those hard to retain compounds using an DS column Enhanced LC/MS sensitivity due to the usage of organic solvent rich mobile phase

71 Retention Mechanism in HILIC Mode Mobile Phase ACN H Uses a reversed phase type mobile phases (Water + rganic Solvent). Hydration Layer H - Si Analyte Si Si Si H Si Hydrogen Bonding Si Si Si Analyte + - Si Si Si Si Si Si Si Si Si Partitioning involves the phase transfer of polar analytes from an organicrich mobile phase into an adsorbed layer of water on the stationary phase. Interaction at the surface occurs through dipoledipole and hydrogen bonding between the surface's stationary phase and polar functional groups of the analyte. Ionic interactions may occur between charged analytes and oppositely charged moieties on the stationary phase or 7

72 7 Comparison Between InertSustain Amide & Conventional DS column As shown below, analytes that are hard to be retained on an DS (C8) can be retained strongly on InertSustain Amide under HILIC mode. The elution order in HILIC is roughly the opposite of that in reversed-phase mode. InertSustain Amide InertSustain C Uracil. Pyridine 3. Phenol HILIC Conditions Column : 5 μm, 50 x 4.6 mm I.D Eluent : A)CH 3 CN B) 0 mm HCNH 4 A/B = 90/0, v/v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 54 nm RP Conditions Column :5 μm, 50 x 4.6 mm I.D Eluent : A) CH 3 B) H A/B = 30/70, v/v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 54 nm

73 Comparison Between InertSustain Amide & Conventional DS column Nucleosides are highly polar which is hard to be retained on an DS (C8) column even under 00 % water mobile phase. As shown below, InertSustain Amide show strong retention of such analytes under HILIC mode. InertSustain Amide InertSustain C8 t0 3 CH 3 CN:0 mm HCNH 4 = 90/0 4 5 t % HC : CH 3 CN =95: t0 5 CH 3 CN:0 mm HCNH 4 = 80/ t % HC in H Conditions Column : 4.6 mm I.D x 50 mm Flow Rate :.0 ml / min Col. Temp. :40 Detection : UV 54 nm Sample:. 5-Methyluridine. Uridine 3. Adenosine 4. Cytidine 5. Guanosine 73

74 74 Comparison Between InertSustain Amide & Conventional DS column Another typical example of InertSustain Amide under HILIC mode showing stronger retention for highly hydrophilic analytes compared to an DS (C8) column. InertSustain Amide InertSustain AQ-C8 T min T min Conditions Column :5 μm, 50 x 4.6 mm I.D Eluent :A) CH 3 CN B) 0 mm HCNH 4 A/B = 90/0, v/v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 5 nm. Cyanulic Acid. Melamine 3. Ammelide 4. Ammeline Conditions Colunn : 5 μm, 50 x 4.6 mm I.D Eluent : 0 mm NaH P 4 :CH 3 CN = 98:, v/v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 5 nm

75 Various Functional Groups for HILIC Columns Inertsil HILIC (Diol Type) InertSustain Amide (Amide Type) InertSustain NH (Amino Type) Si R C C Si R C NH Si R NH Neutral functional group Rapid equilibration time Fast analysis time due to weak retention No retention to acidic compounds Neutral functional group Rapid equilibration time Strong retentivity Basic functional group Prevents anomer resolution for Sugars Silica Column (No Functional Groups) Covalently Bonded, Highly Polar Zwitterionic Functional Group Column Si - Unstable analysis Dissociation occurs at neutral ranges, results in adsorption of peaks by ion exchange interaction No retention to acidic compounds Si R R N + R 4 - S 3 R 3 R Si N + - R R 4 P 4 R 5 R 3 Long equilibration time Buffer compatible 75

76 76 Physical Properties InertSustain Amide Silica Particle Size Surface Area Pore Size Pore Volume Bonded Phase End-capping Carbon Loading ph Range USP Code :ES (Evolved Surface) Silica Gel :3 μm, 5 μm :350 m /g :00 Å (0 nm) :0.85 ml/g :Carbamoyl Groups :None :5 % :~8.5 :L68

77 Comparison Between InertSustain Amide & ther HILIC columns As compared below, the retentivity of InertSustain Amide is similar to XBridge BEH Amide and TSKgel Amide-80. Sample no. 4 (Uridine) was used to determine the retentivity for hydrophilic analytes. InertSustain Amide TSKgel Amide-80 XBridge BEH Amide Atlantis Silica HILIC The retention of basic analyte, Cytosine was strong due to the residual silanols on the silica, which are negatively charged Conditions Column : 5 μm, 50 x. mm I.D (TSKgel Amide-80は.0 mm I.D.) Eluent : A) CH 3 CN B) 0 mm HCNH 4 A/B = 90/0, v/v Flow Rate :0. ml / min Col. Temp. :40 Detection :UV 54 nm Sample :. Toluene. Thymine 3. Urasil 4. Uridine 5. Adenosine 6. Cytosine 7. Ctydine 8. Guanosine 77

78 Melamine and Cyanuric Acid

79 Method Development on HILIC Mode Prefer increasing the retentivity! Area = 5.e 4 Column Eluent : Inertsil DS-4 : A) CH 3 CN B) mm Ammonium Acetate A/B = 5/95, v/v Analytical Conditions Flow Rate : 0.3 ml/min Col. Temp. : 40 Detection : LC/MS/MS (4000 Q TRAP : ESI, Positive, MRM) Injection Vol. : μl Analyte : Melamine μg/l NH N N H N N NH

80 Method Development on HILIC Mode Area =.9e 4 Decrease in sensitivity due to ion suppression Column Eluent : Inertsil DS-4 : A) CH 3 CN B) mm Perfluorooctanoic Acid A/B = 7/73, v/v Addition of an Ion-Pair Reagent Prefer increasing the retentivity! Area = 5.e 4 Column Eluent : Inertsil DS-4 : A) CH 3 CN B) mm Ammonium Acetate A/B = 5/95, v/v Analytical Conditions Flow Rate : 0.3 ml/min Col. Temp. : 40 Detection : LC/MS/MS (4000 Q TRAP : ESI, Positive, MRM) Injection Vol. : μl Analyte : Melamine μg/l NH N N H N N NH

81 Method Development on HILIC Mode Area =.9e 4 Decrease in sensitivity due to ion suppression Column Eluent : Inertsil DS-4 : A) CH 3 CN B) mm Perfluorooctanoic Acid A/B = 7/73, v/v Addition of an Ion-Pair Reagent Prefer increasing the retentivity! Area = 5.e 4 Column Eluent HILIC Mode : Inertsil DS-4 : A) CH 3 CN B) mm Ammonium Acetate A/B = 5/95, v/v Area =.6e 5 Increase in sensitivity due to high organic concentration Column Eluent : Inertsil HILIC : A) CH 3 CN B) mm Ammonium Acetate A/B = 90/0, v/v Analytical Conditions Flow Rate : 0.3 ml/min Col. Temp. : 40 Detection : LC/MS/MS (4000 Q TRAP : ESI, Positive, MRM) Injection Vol.: μl Analyte : Melamine μg/l NH N N H N N NH

82 Which rganic Solvent is the best for HILIC? Difference of Retentivity between rganic Solvents R.T. (min) Acetone Acetonitrile THF -Propanol -Propanol Ethanol Analytical Conditions System :LC Q Trap Column : Inertsil HILIC 3μm 00x. mm Eluent : A) 0 mm CH 3 CNH 4 B) rganic Solvent Flow rate : 0. ml/min. Col. Temp. : 40 Detection : LC/MS/MS ESI,MRM (Positive) Injection Vol. : μl Sample : Melamine (00 μg/l) NH 4 Methanol N N H N N NH Conc. (%)

83 What is the Retention Behavior of HILIC? 83 HILIC Mode 95% CH 3 CN 85% CH 3 CN 75% CH 3 CN 50% CH 3 CN 5% CH 3 CN 5% CH 3 CN Analytical Conditions: Column : Inertsil HILIC 5 μm, 50 x 3.0 mm I.D. Eluent : A) CH 3 CN B) H Flow Rate : 0.4 ml/min Col. Temp : 40 Detection : 0 nm Injection Vol. : 0 μl Sample :. Allantoin 00 mg/l Reversed Phase Mode 5% CH 3 CN

84 Do's and Don'ts for HILIC Columns 84. The best organic solvent is Acetonitrile.. Do not decrease the concentration of Acetonitrile less than 70 %. 3. When adding buffer, Ammonium Acetate should be the best choice as it dissolves easily by Acetonitrile.

85 85 Benefits of InertSustain Amide Higher Hydrophilicity The Amide groups offer strong hydrogen bonding resulting in providing stronger retention of polar analytes compared to Diol groups columns. A Neutral Stationary Phase offer Easy-to-Use & Handle Method Development Unlike silica, amino or zwitterionic stationary phases, a neutral stationary phase do not have an electrical charge which prevents secondary interaction between analytes. ffer faster column equilibration time. Higher chemical stability. High Durability The usage of a radically new type of silica (ES silica), in which the surface of the silica is uniquely modified, enabling precise control of the silica properties provides higher durability compared to regular silica-gels.

86 86 HILIC Mode Your First Choice Column for HILIC mode For Highly Polar Analytes InertSustain Amide

87 Contents. How to Select an HPLC Column How to select your first choice column. How to Select an HPLC Column For Highly Polar Analytes Your first choice column via Reversed Phase Mode Your first choice column via HILIC Mode 3. How to Select an HPLC Column 3 For High-Throughput Analysis, UHPLC Methods Your first choice low carbon loading column 87

88 88 Rapid Analysis using a C8 Phase Samples can be eluted fast by using a C8 phase having a low carbon load %. InertSustainSwift C8 (Carbon Load 9%) Conditions Column Size : 3 μm, 50 x. mm I.D. Eluent : CH 3 /H = 80/0, v/v Flow Rate : 0.3 ml/min Col. Temp. : 40 Detection : UV 54 nm Sample : : Uracil : Toluene 3: Ethylbenzene 4: Propylbenzene 5: n-butylbenzene 6: n-amylbenzene InertSustain C8 (Carbon Load 4%)

89 89 Rapid Analysis, InertSustainSwift C8 Silica : Newly Developed ES Silica Gel Particle Size :.9 μm, 3 μm, 5 μm Surface Area : 00 m/g Pore Size : 00 Å (0 nm) Pore Volume :.00 ml/g Bonded Phase : ctadecyl Groups End-capping : Complete Carbon Loading : 9.0 % USP Code : L ph Range :.0 to 0.0

90 Benefits of InertSustainSwift C8 To Achieve Total Rapid Analysis Rapid Elution of Samples Short Column Equilibration Time Highly Inert Packing Material (Less Tailing of Peaks)

91 Rapid Elution of Samples Conditions Eluent : A) CH 3 B) H A/B = 80/0, v/v : 3 μm, 50 x.mm I.D. : 0.3 ml/min Column Size Flow Rate Col. Temp. : 40 Detection : UV 54 nm Sample :. Uracil. Toluene 3. Ethylbenzene 4. Propylbenzene 5. n-butylbenzene 6. n-amylbenzene InertSustainSwift C8 (3 μm) InertSustain C8(3 μm) 9 9

92 Rapid Elution of Samples Conditions Eluent InertSustainSwift C InertSustain C Column Size Flow Rate Col. Temp. Detection Sample : A) CH3 B) H A/B = 80/0, v/v : 3 μm, 50 x.mm I.D. : 0.3 ml/min : 40 : UV 54 nm. Uracil. Toluene 3. Ethylbenzene 4. Propylbenzene 5. n-butylbenzene 6. n-amylbenzene Inertil DS-SP Inertil DS InertSustain C8 Inertsil DS

93 Rapid Elution of Samples Conditions Eluent : A) CH 3 B) H A/B = 30/70, v/v : 50 x. mm I.D. : 0.4 ml/min Column Size Flow Rate Col. Temp. : 40 Detection : UV 54 nm Sample :. Pyridine. Phenol N:6500 N: MPa N:300 N: MPa InertSustainSwift C8 (.9 μm) Acquity BEH (.7 μm)

94 Rapid Elution of Samples InertSustainSwift C8 Conditions Column Size : 3 μm, 50 x. mm I.D. Eluent : CH 3 /H = 80/0, v/v Flow Rate : 0.3 ml/min Col. Temp. : 40 Detection Sample : UV 54 nm : : Uracil : Toluene 3: Ethylbenzene 4: Propylbenzene 5: n-butylbenzene 6: n-amylbenzene InertSustain C The retentivity of InertSustainSwift C8 is weaker even comparing with a C8 column, which makes it ideal for applications requiring maximum efficiency and throughput. InertSustain C

95 Rapid Elution of Samples High Carbon Load C8 Column (Ex: 0%) Hydrophilic Analyte Hydrophobic Contaminants Conditions Column Size Eluent Flow Rate : 3 μm, 50 x.mm I.D. Col. Temp. : 40 Detection : UV 5 nm Injection Vol : 5 μl Sample :. Etylbenzene : A) CH 3 CN B) 0. % HC,50 mm HCNH 4 in H A/B = 65/35, v/v : 0.3 ml/min Hydrophobic Contaminants InertSustainSwift C8 is optimally bonded to retain hydrophilic compounds without excessive retention of hydrophobic compounds, achieving separations faster than ever before. InertSustainSwift C

96 Benefits of InertSustainSwift C8 To Achieve Total Rapid Analysis Rapid Elution of Samples Short Column Equilibration Time Highly Inert Packing Material (Less Tailing of Peaks)

97 Column Equilibration Time What happens when the column is NT equilibrated enough in gradient methods???. Irreproducible retention time of analytes, especially on highly water-soluble analytes. Insufficient equilibration of column creates slight changes in the mobile phase composition. This results in influencing the reproducibility of retention time.. Generates baseline noise. The baseline noise generates when the solvent inside the column is not equilibrated enough. Fully Equilibrated Insufficient Equilibration

98 Column Equilibration Time What happens when the column is NT equilibrated enough in gradient methods??? 3. Lower sensitivity in LC-MS and LC-MS/MS applications. Insufficient equilibration of column causes changes in ionization efficiency in MS, resulting in variation in sensitivity. Fully Equilibrated Insufficient Equilibration

99 Concentration of rganic Solvent (%) Column Equilibration Test. Methamidophos. Acephate Testing Conditions Column Size: 3 um, 50 mm x. mm I.D. Eluent A): H Eluent B): Acetonitrile Gradient: A/B=95/5 (8 minutes hold) 5 minutes 0/00 5 minutes 0/00 0. minutes 95/5 Equilibration Time, v/v Flow Rate: 0. ml/min Column Temp.: 40 C Detection: UV, 5 nm Sample:. Methamidophos. Acephate 00 Equilibration Time 5 minutes 0 minutes 5 minutes 0 minutes

100 Results of Column Equilibration Test InertSustainSwift C8 XBridge C8 Results when the column is fully equilibrated Equilibration for 5 min Equilibration nearly completed Unstable baseline Equilibration for 0 min Equilibration completed Equilibration completed Fast equilibration of column leads to shorter total operation time

101 Stability Under 00 % Aqueous Mobile Phases When analyzing hydrophilic compounds under water rich mobile phase condition, once the pump is stopped, the hydrophobic bonded group pushes the aqueous mobile phase out off the pore in an irreversible fashion, in what has become known as the dewetting phenomenon. High rganic Concentration Adding flow with high pressure Analyzing with 00% Aqueous Eluent Stop Flow (Pressure Release) Dewetting Pore DS Groups The pore can be generated or conditioned by introducing Methanol or Acetonitrile. The DS groups pushes the aqueous mobile phase out off the pore in an irreversible fashion, results in loss of retention of sample

102 Stability Under 00 % Aqueous Mobile Phases First Injection Testing Conditions ) 00 % water is introduced into column over 60 minutes. ) Conduct analysis. 3) Stop flow for 5 minutes. 4) 00 % water is introduced again into column over 30 minutes. 5) Stop flow for 5 minutes again. 6) Conduct analysis Second Injection Variation: 99.6 (%) Conditions Column : InertSustainSwift C8 (5 μm, 4.6 x 50) Eluent : 00 % H Flow rate :.0 ml/min Col. Temp.: 40 Detection : UV 54 nm Sample :.Cytosine.Uracil 3.Guanine 4.Thymine 5.Adenine Third Injection Variation: 99.8 (%) InertSustainSwift C8 demonstrates excellent resistance to dewetting guaranteeing highly stable, reliable and reproducible chromatograms for gradient methods

103 Benefits of InertSustainSwift C8 To Achieve Total Rapid Analysis Rapid Elution of Samples Short Column Equilibration Time Highly Inert Packing Material (Less Tailing of Peaks)

104 Benefits of Highly Inert Packing Material 3 InertSustainSwift C8 (.9 um, 50 x. mm I.D.) L column DS ( um, 50 x. mm I.D.) 5 Conditions Eluent Flow Rate Col. Temp. : 40 Detection : UV 30 nm Injection Vol : 0.5 μl Sample : A) CH 3 CN B) 5mM K HP 4 (ph 7.0, KH P 4 ) A/B = 30/70,v/v : 0.4 ml/min : :Uracil :Pyridine 3:Phenol 4:Berberine chloride 5:Dextromethorphan InertSustainSwift C8 employs a highly inert packing material which provides pure hydrophobic interaction between analytes without generating secondary interaction between residual silanols delivering rapid results. Poorly end-capped columns often show tailing of peaks due to the presence of silanols resulting in longer analysis time

105 Benefits of Highly Inert Packing Material

106 Benefits of Highly Inert Packing Material Reproducibility You Can Rely n Conditions Column : 3 um, 50 x 4.6 mm I.D. Highly Basic. Dextromethorphan Highly Acidic. Brilliant Blue FCF Highly Chelating. Hinokitiol Tf =.38 Tf =.38 Tf =.44 LT LT Tf =.33 Tf =.45 Tf =.48 LT 3 Tf =. Tf =.3 Tf =

107 Benefits of Highly Inert Packing Material Sub μm μm 0 Time 0 (min) 0 Time(min) μm Conditions Eluent Flow Rate : Col. Temp. : 40 Detection : UV 0 nm Injection Vol : Sample: : A) CH 3 CN B) 5mM K HP 4 (ph 7.0, KH P 4 ) A/B = 60/40,v/v. Chlorpheniramine. Triprolidine 3. Homochlorcyclizine 4. Hydroxyzine 5. Clemastine Sub μm (. 50) : 0.4 ml/min : 0.5 μl 3 μm (. 50) : 0.3 ml/min : μl 5 μm (4.6 50) :.0 ml/min : μl InertSustainSwift C Zorbax Eclipse Plus C8 InertSustainSwift C8 demonstrates excellent reproducibility between various particle size columns

108 Benefits of Highly Inert Packing Material Ion suppression in liquid chromatography coupled with mass spectrometry (LC MS, LC-MS/MS) can occur when a co-eluted compound suppresses the ionization of the sample molecules in a mass spectrometer s source. Cotaminants Ion Suppression The usage of poorly end-capped columns have a potential for causing ion suppression due to the presence of silanols leading to tailing peaks of contaminants which may co-elute with the analyte of interest. Target Analyte Low Sensitivity Poorly Inert Packing Material (Poorly End-capped Column) As a result, the effects on the important analytical parameters including precision, accuracy and limit of detection (sensitivity) can be extensive, severely limiting the validity of an assay's results

109 Benefits of Highly Inert Packing Material Achieving Separation & Preventing Ion Suppression Cotaminants Target Analyte Higher Sensitivity Highly Inert Packing Material (Highly End-capped Column) The usage of highly end-capped columns not only have a low potential for causing ion suppression, but also can deliver higher sensitivity with more reliable accurate analysis

110 Benefits of Highly Inert Packing Material 5.0 (x,000,000) :37.30(+) Analysis of Basic Drug under Acidic (Formic Acid) Mobile Phase Conditions Procaineamide: Q pg Conditions System Column Eluent : Nexera LCMS-8030 plus : InertSustainSwift C8 (3 μm, 50 x. mm I.D.) : A) 0.% HC in H B) 0.% HC in CH 3 CN A/B = 95/5-5 min - 0/00-5min - 0/00 0.min - 95/5-5min - 95/5 -,v/v : 0. ml/min Flow Rate Col. Temp. : 40 Detection : LC/MS (ESI) min Tailing of peaks or adsorption of peaks can be experienced when a column has residual adsorption sites. Such phenomenon would not be observed on InertSustainSwift C8 as the silanols are completely end-capped, having a neutral silica surface even under such mobile phase conditions

111 Benefits of Highly Inert Packing Material Effects of Buffer Concentration (Ammonium Acetate) on Peak Shapes using Basic Drugs N CH 3 CH 3 N N CH 3 Dibucaine (pka 8.5) CH 3 N CH 3 CH 3 N S Promethazine (pka 9.) Conditions System : Nexera LCMS-8030 plus Column size : (3 μm, 50. mm I.D.) Eluent : A) Ammonium Acetate in H B) Ammonium Acetate in CH 3 :A/B = 30/70,v/v Flow Rate : 0. ml/min Col. Temp. : 40 Detection : LC/MS/MS (ESI, Positive, MRM) Sample : Each 0. (mg / ml) Q > Q3 Dibcaine : > 7.5 (+) Promethazine : > 86.5 (+)

112 Benefits of Highly Inert Packing Material Effects of Buffer Concentration (Ammonium Acetate) on Peak Shapes using Basic Drugs. Dibucaine (pka 8.5). Promethazine (pka 9.) mm 5 mm 0 mm (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min Tailing (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min CAPCELLPAK MG lll C8

113 Benefits of Highly Inert Packing Material Effects of Buffer Concentration (Ammonium Acetate) on Peak Shapes using Basic Drugs. Dibucaine (pka 8.5). Promethazine (pka 9.) mm 5 mm 0 mm (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min Tailing (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min Hypersil GLD C

114 Benefits of Highly Inert Packing Material Effects of Buffer Concentration (Ammonium Acetate) on Peak Shapes using Basic Drugs. Dibucaine (pka 8.5). Promethazine (pka 9.) mm 5 mm 0 mm (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min Decrease in Sensitivity Zorbax Eclipse Plus C

115 Benefits of Highly Inert Packing Material Effects of Buffer Concentration (Ammonium Acetate) on Peak Shapes using Basic Drugs. Dibucaine (pka 8.5). Promethazine (pka 9.) mm 5 mm 0 mm (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min (x00,000) 3.0 4:345.0>7.5(+) 7:87.05>86.5(+) min InertSustainSwift C8 As shown above, InertSustainSwift C8 maintains the same level of sensitivity and sharp peak shapes between various buffer concentration

116 Another Value-Added Benefit, Low Column Bleed HPLC column bleed is a major source of background signal in LC-MS, LC-MS/MS analyses. This phase bleed occurs when the bonded phase or unreacted reagent elutes from the column during the analysis. Generally, highly end-capped columns often show high column bleed in LC-MS, LC-MS/MS applications (x,000,000,000) TIC(+)@ YMC-Triart C8 Blank InertSustainSwift C8 Conditions System : Nexera LCMS-8030 plus Column Size : 3 μm, 50. mm I.D.) Eluent : A) 0. % HC in H B) 0. % HC in CH 3 CN A/B = 95/5-5 min - 0/00 5 min 0/00 0. min - 95/5 5 min - 95/5,v/v Flow Rate : 0. ml/min Col. Temp. : 40 Detection : LC/MS (ESI, Positive, Mass Range: ) Q SCAN : m/z min As tested above, not only InertSustainSwift C8 is a highly end-capped column, but also provides low column bleed which makes it compatible for LC-MS, LC-MS/MS applications

117 Another Value-Added Benefit, Low Column Bleed Comparison of Column Bleed (x,000,000) :38.5(+) ther Brand s C8 Column.Procaine: Q (x,000,000) :38.5(+) InertSustainSwift C8 Higher Sensitivity Column Bleed min (x,000,000) :339.55(+).Papaverine: Q min min 6.0 (x00,000) :339.55(+) min 7

118 Applications Analysis of Beta Blockers in 60 Seconds (x,000,000) 3:337.0>6.05(+) 4:67.5>45.00(+) 8:39.00>6.95(+) 9:30.05>54.00(+) 0:49.80>6.00(+) min 5 Conditions System Column Eluent : Nexera LCMS-8030 plus :.9 μm,. x 50 mm : A) 0 mm Ammonium Formate in H B) 0 mm Ammonium Formate in CH 3 A/B = 70/ min - 40/60 0.5min - 0/00 0.min 0/00 0.0min -70/30 0.5min - 70/30,v/v : 0.6 ml/min Flow Rate Col. Temp. : 40 Detection : LC/MS/MS (ESI, Positive, Negative MRM) Sample : Each 00 (μg/l) Q > Q3. Acebutolol : > 6.05 (+). Atenolol : 67.5 > (+) 3. Labetalol : > 6.95 (+) 4. Nadolol : > (+) 5. Pindolol : > 6.00 (+) InertSustainSwift C8.9 μm, 50 mm x. mm I.D

119 Applications Analysis of Tulathromycin A on Animal Products fficial Method from the Japanese Health, Labour and Welfare Ministry.50 (x0,000) :806.60>577.35(+) pg min InertSustainSwift C8 3 μm,50 mm x. mm I.D. Conditions System : LC-MS or LC/MS/MS Column : 3 μm,. x 50 mm Eluent : A) Acetonitrile B) Trifluoroacetic acid aqueous solution A/B = 5/75,v/v Flow Rate : Analyte to be eluted from 4 to 6 minutes Col. Temp. : 40 Detection : LC/MS/MS (ESI, Positive) m/z: 577, 807 NH CH 3 H H 3 C H CH 3 H H CH 3 CH 3 H 3 C H N CH 3 H CH 3 H CH 3 H H CH 3 H 3 C CH 3 N H CH 3. Tulathromycin A (MW ) 9 9

120 Applications Column Eluent Analysis of Terbinafine Chloride (Under the Condition of the Japanese Pharmacopoeia) : 4.0 mm I.D. x 5 mm length, SUS, 3 μm, DS Phase : Tetramethylammonium Hydroxide aqeuous solution, ph adjusted at 8.0 by Phosphoric Acid/Acetonitrile/ THF = 40/40/0 Col. Temp. : To be set around 5 constantly Flow Rate : Adjust the flow rate to make Terbinafine elute around 8.5 minutes System Suitability Test Prepare 40 mg of Terbinafine Hydrochloride and 3.5 mg of p-terphenyl and dissolve by 00 ml Methanol. Inject 0 ul of this sample solution and analyze by referring to the above conditions. p-terphenyl shall be eluted first, then Terbinafine Chloride. Resolution: More than 6.0 between Terbinafine Hydrochloride and p-terphenyl. H 3 C CH 3 H 3 C CH 3 N HCl. Terbinafine Chloride 0 0

121 Applications Analysis of Terbinafine Chloride (Under the Condition of the Japanese Pharmacopoeia) InertSustainSwift C8.46 ml/min. p-terphenyl 00 mg/l. Terbinafine Chloride 7.5 mg/l InertSustain C

122 Summary To Achieve Total Rapid Analysis Rapid Elution of Samples Short Column Equilibration Time Highly Inert Packing Material (Less Tailing of Peaks)

123 Summary InertSustainSwift C8 is a perfect column for the following applications due to it s benefits explained earlier.. Dissolution Test. Continent Uniformity Test 3. Assay Test 4. LC-MS, LC-MS/MS Researchers 5. Bioequivalence Test 6. Peptide samples (M.W. 5,000 to 0,000) 3 3

124 4 Contents 4. How to Select an HPLC Column 4 For Separation of Basic Analytes & its Related Substances, Process Impurities Your first choice high carbon loading column 5. How to Select an HPLC Column 5 ther stationary phases that may improve separation

125 Inertsil DS-HL Physical Properties Silica Particle Size Surface Area Pore Size Pore Volume Bonded Phase End-capping Carbon Loading ph Range USP Code :3 Series High Purity Silica Gel :3 μm, 5 μm :450 m /g :00 Å (0 nm) :.05 ml/g :ctadecyl Groups :Yes :3 % :~7.5 :L * HL : High Carbon Load = 3 % 5

126 Benefits of Inertsil DS-HL High Selectivity ffering unique separation pattern which a conventional C8 columns does not offer. Ultra High Retentivity Strong separation power compared to conventional C8 columns. Provides strong retentivity even under organic solvent rich mobile phases. High Inertness Delivers sharp peaks for bases and acids. 6

127 Retention Characteristics of Inertsil DS-HL As shown from the following image, Inertsil DS-HL offer both high selectivity and strong hydrophobic interaction. The Inertsil DS-HL columns are fully endcapped which deliver sharp peaks for bases and acids. * The following plot may not be representative of all phases in each category and for all analytes. Inertsil DS-EP Red Green Black : Non-endcapped columns : General end-capped columns : Highly end-capped columns Steric Selectivity Ph columns C8 columns InertSustainSwift C8 Inertsil DS-P Inertsil DS-4 Conventional DS columns Inertsil DS-3 InertSustain C8 Inertsil DS-HL Retentivity 7

128 Retention Characteristics of Inertsil DS-HL *Steric Selectivity * ratio of k-values for triphenylene and o-terphenyl Inertsil WP300 C4 Inertsil C4 Inertsil WP300 C8 InertSustainSwift C8 Inertsil C8-4 Inertsil C30 S-Select Inertsil WP300 C8 InertSustainSwift C8 Inertsil DS-SP Inertsil DS Inertsil C8-3 InertSustain C8 Inertsil DS-4 Inertsil DS-EP Inertsil DS-P Inertsil DS Retentivity (Amylbenzene k ) Inertsil DS-80A Inertsil DS-3 InertSustain C8 InertSustain AQ-C8 Inertsil DS-HL show very different characteristics compared to other columns available in the market Inertsil DS-HL 8

129 Distinctive Separation Pattern via Inertsil DS-HL Hydrophobicity is the primary mechanism of analyte interaction with conventional C8 columns. For a given phase, retention time is proportional to the hydrophobicity of the molecule. Therefore, the separation of structurally similar analytes are often difficult to be achieved on conventional C8 columns. The densely bonded C8 groups on Inertsil DS-HL creates superior planar recognition enabling complete separation for such analytes. Inertsil DS-HL (Carbon Loading 3 %) InertSustain C8 (Carbon Loading 4 %),3 Unseparated Peaks 3 Complete Separation Conditions Colμmn size : 5 μm, 50 x 4.6 mm I.D. Eluent : A) CH 3 CN B) H A/B = 85/5, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm. o-terphenyl. m-terphenyl 3. p-terphenyl 9

130 Distinctive Separation Pattern via Inertsil DS-HL Stereoselectivity is indicated by o-terphenyl and Triphenylene. -Terphenyl has a twisted tertiary structure and Triphenylene has a planar structure. Inertsil DS-HL elutes Triphenylene later against o-terphenyl which proves the stereoselectivity to be very high. Inertsil DS-HL not only provide high retentivity, but with high stereoselectivity. Selectivity Test o-terphenyl (Non-planar) Triphenyl (Planar) Inertsil DS-HL Separation Factor tri/o-ter InertSustain C8 (Conventional C8) Separation Factor tri/o-ter YMC-Triart C8 ExRS (High Carbon Loading C8) Conditions Colμmn size : 5 μm, mm I.D. Flow Rate :.0 ml/min.uracil Eluent : A) CH 3 Col. Temp. : 40.Caffeine 3.Phenol B) H Detection : UV 54 nm 4.n-Butylbenzene A/B = 80/0, v / v Separation Factory.63 5.n-Amylbenze 6.o-Terphenyl 7.Triphenylene tri/o-ter 30

131 Ultra High Retentivity of Inertsil DS-HL The higher the hydrophobicity of analyte, stronger the retention on Inertsil DS-HL columns. For the retention of hydrophilic analytes, InertSustain AQ-C8 columns are appropriate. * The following plot may not be representative of all phases in each category and for all analytes. Image of Retention Characteristic The range of structural analogs where improved separation can be expected Inertsil DS-HL InertSustain AQ-C8 Retentivity Hydrophobic Interaction InertSustain C8 InertSustainSwift C8 Hydrophilicity Mobile phase: water rich Hydrophobicity of Analyte Hydrophobicity Mobile phase: organic rich 3

132 Ultra High Retentivity of Inertsil DS-HL The higher the hydrophobicity of analyte, stronger the retention on Inertsil DS-HL columns. Inertsil DS-HL columns strongly retain highly hydrophobic analytes compared to conventional DS columns. Retentivity () (Hydrophobic analytes) Inertsil DS-HL Acetophenone (Hydrophobicity : Low) Naphthalene (Hydrophobicity : Moderate) Conditions Colμmn Size : 5 μm, 50 x 4.6 mm I.D. Eluent : A) CH 3 CN B) H A/B = 65/35, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm Acetophenone.Benzene 3.Toluene 4. Naphthalene InertSustain AQ-C InertSustain C8 (Conventional DS) InertSustainSwift C

133 Ultra High Retentivity of Inertsil DS-HL For the analysis of highly polar analytes under water rich mobile phases, InertSustain AQ-C8 columns provide stronger retention. Under 00 % water mobile phase, InertSustain AQ-C8 columns are highly recommended as it delivers stronger retention of highly polar analytes. Inertsil DS-HL may cause dewetting phenomenon under such mobile phase. Retentivity () (Highly Polar analytes) Inertsil DS-HL Conditions Colμmn size : 5 μm, 50 x 4.6 mm I.D. Eluent: H (00 %) Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm Sample:.Cytosine.Uracil 3.Thymine InertSustain AQ-C InertSustain C8 (Conventional DS) InertSustainSwift C

134 Ultra High Retentivity of Inertsil DS-HL As shown below, Inertsil DS-HL can be used under organic solvent rich mobile phases without sacrificing retentivity. Retentivity (3-) (Under various Methanol Concentration) Inertsil DS-HL (90 % Methanol) Conditions Colμmn size : 5 μm, 50 x 4.6 mmi.d. Eluent : A) CH 3 B) H Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm.uracil.caffeine 3.Phenol 4.n-Butylbenzene 5. n-amylbenze 6. o-terphenyl 7. Triphenylene InertSustainSwift C8 (80 % Methanol) XBridge C8 (80 % Methanol)

135 Ultra High Retentivity of Inertsil DS-HL As shown below, Inertsil DS-HL can be used under organic solvent rich mobile phases without sacrificing retentivity. Retentivity (3-) (Under various Methanol Concentration) Inertsil DS-HL (85 % Methanol) Conditions Colμmn size : 5 μm, 50 x 4.6 mmi.d. Eluent : A) CH 3 B) H Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm.uracil.caffeine 3.Phenol 4.n-Butylbenzene 5. n-amylbenze 6. o-terphenyl 7. Triphenylene Inertsil DS-4 (80 % Methanol) L-column DS (80 % Methanol)

136 High Inertness Commercially available high carbon loading DS columns often show severe tailing of peaks due to the insufficient end-capping of free silanols. Inertsil DS- HL columns offer superior peak shapes for not only for bases but for acids as well due to the high-end end-capping technology from GL Sciences. Basic Compound Test Acidic Compound Test Conditions Colμmn Eluent Flow Rate Col.Temp. :40 Detection :UV 30 nm :5 μm, mm I.D. Sample: :A) CH 3 CN B) 5 mm K HP 4 (ph 7.0) A/B = 30/70, v/v :.0 ml/min. Uracil. Pyridine 3. Phenol 4. Berberine Conditions Colμmn Eluent :5 μm, mm I.D. :A) CH 3 CN B) 0.% H 3 P 4 A/B = 5/75, v/v :.0 ml/min Flow Rate Col.Temp. :40 Detection :UV 30 nm Sample:. Uracil. Phenol 3. Salicylic acid 36

137 High Inertness Inertsil DS-HL provide sharp peak shape for basic analytes, while Inertsil DS-P show adsorption to the packing material as it is a non-endcapped column. Inertsil DS-HL (Carbon Loading 3 %) Conditions Column size : 5 μm, 50 x 4.6 mm I.D. Eluent : A) CH 3 B) H A / B = 30/70, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm Sample:.Pyridine.Phenol Inertsil DS-P (Carbon Loading 9 %) : Adsorption observed InertSutain C8 (Carbon Loading 4 %)

138 High Inertness Inertsil DS-HL provide sharp peak shape for strong basic analytes as well, while other column brands failed. Inertsil DS-HL (Carbon Loading 3 %) Symmetry Factor :.8 Conditions Column size : 5 μm, 50 x 4.6 mm I.D. Eluent : A) CH 3 CN B) 5mM potassium phosphate (ph7.0) A/B = 30/70, v/v Flow Rate:.0 ml/min Col. Temp.: 40 Detection: UV 30 nm Sample;. Pyridine. Berberine YMC-Triart C8 ExRS (Carbon Loading 5 %) Symmetry Factor : Develosil HSR C8 (Carbon Loading %) Symmetry Factor :

139 % Initial 保持時間維持率 Retention % Time (%) High Durability % 理論段数維持率 Initial (N) (%) As shown below, Inertsil DS-HL maintained high durability as InertSustain C8 and Inertsil DS-4. Low ph Resistance Test 0 保持時間維持率 (%) 0 理論段数維持率 %(%) % InertSustain C8 Inertsil DS-3 Inertsil DS-4 Inertsil DS-HL 70 InertSustain C8 Inertsil DS-3 Inertsil DS-4 Inertsil DS-HL Hours of 時間 Purging (h) (h) 時間 (h) Hours of Purging (h) Storing condition in the oven at 60 C : CH 3 CN/H /TFA(0/90/), ph= 39

140 保持時間維持率 (%) % Initial Retention % Time High Durability % 理論段数維持率 Initial (N) % (%) As shown below, Inertsil DS-HL maintained high durability as Inertsil DS-4 and Inertsil DS-3. High ph Resistance Test 保持時間維持率 (%) 0 保持時間維持率 (%) 0 段数維持率 (%) 理論段数維持率 (%) InertSustain C8 Inertsil DS-3 40 InertSustain C8 Inertsil DS-3 0 Inertsil DS-4 Inertsil DS-HL 0 Inertsil DS-4 Inertsil DS-HL 時間 (h) Hours of Purging (h) 時間 (h) Hours of Purging (h) Purging solvent : 50mM TEA (ph 0.0) / CH3 (70/30), 50 C at.0 ml/min 40

141 Low Bleed for LC-MS Compatibility Intensity, cps 4.e9 4.0e9 3.8e9 3.6e9 3.4e9 3.e9 3.0e9.8e9.6e9.4e9.e9.0e9.8e9.6e9.4e9.e9.0e9 8.0e8 6.0e8 4.0e8.0e8 0.0 Conditions Column :Inertsil DS-HL( 3 μm,50. mm I.D. ) InertSustainSwift C8( 3 μm,50. mm I.D. ) Eluent :A)0. %HC-H B)0. %HC-CH 3 CN A/B=95/5-0min-5/95-0.min-95/5-0min gradient Flow Rate :00 μl/min Col.Temp. :40 Detection :MS ( API3000;ESI,Pos.,Q MS Scan ) Da Injection Vol. : μl Sample :Eluent A Blue Red Green : System blank : Inertsil DS-HL : InertSustainSwift C Time, min 4

142 Sample Loading Capacity Comparison of Sample Loading Capacity Sample Preparation Alkylphenol C~4.4-Ethylphenol.p-n-Propylphenol 3.4-n-Butylphenol : 0.5 mol/l : mol/l : 0.5 mol/l LC conditions Column : Inertsil DS-HL 5 μm, 4.6 x 50 mm XBridge C8 5 μm, 4.6 x 50 mm Eluent : A)H, B)CH 3 A/B=30/70-5 min-0/00-0. min-30/70-0 min-30/70 Flow Rate :.0 ml/min Col.Temp. : 40 Detection : 300 nm Injection Vol. :,5,0,5,50,75,00,5 μl 4

143 N (Propyl benzene Sample Loading Capacity Relative value of N (Propyl benzene) Concentrated standards were injected with various injection volumes separately to evaluate the sample loading capacity. The efficiency (N) decreases as the injection volume increases. However, Inertsil DS-HL is less affected by the increase in injection volumes due to the high surface area of silica gel with the C8 groups densely bonded. (Inertsil DS-HL, Carbon Loading : 3 %, XBridge C8, Carbon Loading : 8 %) Comparison of Sample Loading Capacity Injection Volume vs N (Efficiency) Inertsil DS-HL XBridge C 試料注入量 Injection Volume (μl)(ul) A plot of relative value when the efficiency (N) is 00 at μl injection volume Inertsil DS-HL XBridge C 試料注入量 Injection Volume (μl) (ul) 43

144 Sample Loading Capacity As proven below, Inertsil DS-HL is optimized for preparative and process scale applications. Comparison of Sample Loading Capacity Inertsil DS-HL XBridge C8 50 μl mvolt μl mvolt μl mvolt Complete separation 00 μl mvolt Incomplete separation

145 Summary of Inertsil DS-HL Alternative Selectivity to Conventional C8 columns Appropriate to use when observing separation issues with standard C8 columns. Perfect for basic molecules & its related substances, process impurities. utstanding Alternative to ther High Carbon Loading C8 columns Ideal selection when observing separation issues or poor peak shapes on other high carbon loading C8 columns. ptimized for Preparative and LC-MS applications For preparative applications, high surface area & carbon load leads to maximum sample loading capacity. For LC-MS applications, retention can be maintained whilst reducing the aqueous content of the mobile phase, thus increasing sensitivity. 45

146 Application : Vitamin D, D3 Structurally similar analytes are difficult to be separated on conventional DS columns. A classic example is illustrated below using Vitamin D and D3. Inertsil DS-HL is an ideal selection for such applications delivering complete separation. Condition Column : 3 μm,. x 50 mm Eluent : CH 3 Flow Rate : 0.3 ml/min Col.Temp. : 5 Detection : 65 nm Injection Vol. : 5 μl Analyte : 5 μg/ml each H H H H H H H H H.Vitamin D (calciferol) H H H H H H H H.Vitamin D 3 (cholecalciferol) Inertsil DS-HL mvolt mvolt Complete Separation InertSustain C8 mvolt mvolt Incomplete separation

147 Application : ctadecenoic Acid Methyl Ester, cis/trans-isomers Cis- and trans-isomers of long-chain fatty acids were injected to evaluate the separation on the following C8 columns. As shown below, although complete separation was not achieved, Inertsil DS-HL delivered the best separation. Inertsil DS-HL Resolution : Condition Colmn size : 5 μm, 4.6 x 50 mm Eluent : CH 3 Flow Rate :.0 ml/min Detection : 05 nm Col.Teml : 30 Injection.Vol. : 0 μl Analyte : 5 mm in CH 3 each InertSustain C L-column DS Resolution : cis9-c8: methyl ester (Methyl leate) XBridge C trans9-c8: methyl ester (Methyl Elaidate) 47

148 Application : Adrenal Cortical Hormones Simultaneous analysis of adrenal cortical hormones were operated using Inertsil DS-HL and conventional C8 column. Inertsil DS-HL achieved complete baseline separation on all 6 analytes. Volts Volts Inertsil DS-HL(3 μm mm) InertSustain C8(3 μm mm) Condition Eluent :A)H B)CH 3 CN A/B=65/35-5 min-6/38-5 min-58/4-5 min -0/90-0. min-65/35-0 min Col.Temp. :30 Flow Rate :0.3 ml/min Detection :60 nm Analyte.Prednisolone.Hydrocortisone 3.Cortisone 4.6-α-Methylprednisolone 5.Betamethasone 6.Dexamethasone 7.Corticosterone 8.Triamcinolone Acetonide 9.Fluocinolone Acetonide 0.Prednisolone Acetate.Hydrocortisone Acetate.Cortisone Acetate 3.Fluocinonide 4.Betamethasone Valerate 5.Deoxycorticosterone 6.Beclomethasone 48

149 Application : Tocotrienols, Tocopherols Highly hydrophobic vitamin E were analyzed under 00 % methanol using Inertsil DS-HL and all compounds were well retained. All compounds were well separated except between β-tocopherol and γ-tocopherol. Inertsil DS-HL Condition Colμmn size :5 μm, 4.6 x 50 mm Eluent :CH 3 Flow Rate :.0 ml/min Detection :0 nm Col.Teml :30 Injection.Vol. :5 μl Analyte :0.0 mg/ml in CH 3 each : δ-tocotrienols : γ-tocotrienols 3: α-tocotrienols 4: δ-tocopherols 5: β-tocopherols 6: γ-tocopherols 7: α-tocopherols

150 InertSustain Cyano Physical Properties Silica Particle Size Surface Area Pore Size Pore Volume Bonded Phase End-capping Carbon Loading ph Range USP Code :Newly Developed ES Silica Gel :3 μm, 5 μm :350 m /g :00 Å (0 nm) :0.85 ml/g :Cyanopropyl Groups :Yes :8 % :~7.5 :L0 50

151 Benefits of InertSustain Cyano Truly Reliable and Reproducible Endlessly reproducible from column-to-column and batch-to-batch. Excellent for Typically All USP L0 Monographs The InertSustain Cyano columns are highly recommended for all pharmacopeia methods requiring a Cyano phase to be used. (Ex: USP L0) High Inertness Delivers sharp peaks for virtually any type of analytes. 5

152 Comparison of Batch-To-Batch Reproducibility As proven below, InertSustain Cyano provide exceptional reproducibility from batch-to-batch even with those challenging strong basic compounds such as Dextromethorphan or Berberine. Batch 3 Batch 3 Conditions Column Eluent Flow Rate Col.Temp. Detection Sample :5 μm,50 x 4.6 mm I.D. :A) CH3CN B) 0.% H3P4 A/B = 5/75, v/v :.0 ml/min :40 :UV 30 nm :. Uracil. Dextromethorphan 3. Berberine Batch Batch Batch 3 Batch InertSustain Cyano ZRBAX SB-CN 5

153 Comparison of Peak Shapes using Strong Basic Compounds As shown below, InertSustain Cyano columns provide symmetric peaks for strong bases and chelating compounds, delivering highly stable chromatograms for qualitative and quantitative analysis. H 3 C N H CH3. Dextromethorphan N CH 3 CH 3 3. Berberine Conditions Column Eluent Flow Rate Col.Temp. Detection Sample :5 μm,50 x 4.6 mm I.D. :A) CH3CN B) 0.% H3P4 A/B = 5/75, v/v :.0 ml/min :40 :UV 30 nm :. Uracil. Dextromethorphan 3. Berberine InertSustain Cyano ZRBAX SB-CN XSelect HSS CN 53

154 Comparison of Peak Shapes using Strong Chelating Compounds As shown below, InertSustain Cyano columns provide symmetric peaks for strong chelating compounds, delivering highly stable chromatograms for qualitative and quantitative analysis. Conditions Column Eluent Flow Rate Col.Temp. Detection Sample : 5 μm, mm I.D. : A) CH 3 CN B) 0.% H 3 P 4 A/B = 5/75, v/v :.0 ml/min : 40 : UV 30 nm :. Hinokitiol.. Hinokitiol InertSustain Cyano ZRBAX SB-CN XSelect HSS CN 54

155 Highly Stable Chemistry for Normal-Phase Separations Although the InertSustain Cyano columns are originally shipped in reversed-phase solvents, it can also be used for normal-phase separations by properly equilibrating the column with ethanol or -propanol prior to the analysis ,3 4 5 A packing down of packing material was observed at the column inlet side Conditions Column Eluent Flow Rate Col.Temp. Detection Sample :5 μm, 50 x 4.6 mm I.D. :A) n-hexane B) Ethanol A/B = 90/0, v/v :.0 ml/min :40 :UV 0 nm. Progesterone. Estrone 3. β-estradiol 4. Corticosterone 5. Hydrocortisone InertSustain Cyano ZRBAX SB-CN XSelect HSS CN 55

156 Applications Analysis of Nortriptyline Hydrochloride Capsules [USP Method] N : 7988 Tailing factor :.0 USP Column: 5 μm, 50 x 4.6 mm I.D. (L0) System suitability requirements: Efficiency (N) : >500 Tailing factor : < 3.0 Sample Conc. : 0.38 mg/ml (in Methanol) Mobile Phase : ACN : CH3 : mm Potassium phosphate (ph 6.7) = 40 : 43 : 7 Flow Rate :.5 ml Detection : UV 39 nm Injection : 5 μl

157 Applications Analysis of Sertraline Hydrochloride [USP Method] USP Column: 5 μm, 50 x 4.6 mm I.D. (L0) System suitability requirements: Tailing factor : <.0 Tailing factor :.4 Sample Conc. : mg/ml (in Mobile Phase) Mobile Phase : CH3 : 0.% (v/v) Phosphoric acid = : Flow Rate :.5 ml Detection : UV 0 nm Colum Temp. : 30 Injection : 0 μl t

158 Applications Analysis of Tetracaine Hydrochloride phthalmic Solution [USP Method] USP Column: 5 μm, 50 x 4.6 mm I.D. (L0) System suitability requirements: Efficiency (N) : >500 Tailineg factor : <.0 N : 9679 Tailing factor :.6 Sample Conc. : 0. mg/ml (in Water) Mobile Phase : ACN : 0 mm Ammonium phosphate (ph 3.0) = 30 : 70 Flow Rate :.0 ml Detection : UV 80 nm Injection : 0 μl t

159 GL Sciences HPLC Column Selection Guide (/) InertSustain C8 First Choice C8 Column InertSustain AQ-C8 Ideal for Maximizing Retention for Highly Polar Compounds in Reversed Phase Methods with Highly Aqueous Mobile Phases InertSustainSwift C8 Rapid Elution of Samples in Isocratic Methods and Rapid Column Equilibration Time in Gradient Methods Inertsil DS-HL Ultra High Retentivity, High-Density Bonding of C8 Phase Ideal for Separation of Basic Molecules & its Related Substances, Process Impurities InertSustain Amide Excellent for those hard to retain compounds using an DS column First choice HILIC column under HILIC Mode Separation 59

160 GL Sciences HPLC Column Selection Guide (/) InertSustain Cyano First Choice USP L0 Column The Most Reliable and Reproducible Cyano Column 60

161 Comparison of rganic Solvents Separation Efficiency 7% CH3CN 80% CH3 56% THF Phenol Caffeine o-terphenyl Butylbenzene Triphenylene Amylbenzene Phenol Caffeine Butylbenzene Amylbenzene o-terphenyl Triphenylene Phenol Caffeine Triphenylene o-terphenyl Butylbenzene Amylbenzene Same instrument, column and sample were used. nly changed the type of an organic solvent. 6

162 Improving Separation using the Mobile Phase Separation/Elution pattern can be different depending on the type of organic solvent. NH NH.p-Methylhippuric Acid.m-Methylhippuric Acid 8% Acetonitrile 30% Methanol 0% -Propanol % THF

163 Improving Separation using the Mobile Phase Analytical Conditions Column : Inertsil DS-3 5 μm, mm I.D. Eluent : rganic solvent / 0.% H 3 P 4 Flow Rate :.0 ml/min Detector : UV 84 nm Col.Temp. : 40 4, 5 Sulfa drugs ) Sulfamerazine ) Furazolidone 3) xolinic acid 4) Sulfadimethoxine 5) Sulfaquinoxaline 6) Nalidixic acid % CH 3 CN 30% CH 3 CN 40% CH 3 63

164 Tips on Method Validation. 64 Validation on Mobile Phases. Make sure the resolution or retention time is NT changing by the adjustment error on the mobile phase. It is strongly recommended to validate the mobile phase especially when adjusting the ph of the mobile phase or using an ion-pair reagent. Regarding validation, the preparation method of the mobile phase shall be documented like an SP.

165 Tips on Method Validation. 65 Pop Quiz!!!. Prepare Acetonitrile. Prepare Buffer 3. Mix Both 4. Finally, set the ph

166 Tips on Method Validation. 66 Pop Quiz!!!. Prepare Acetonitrile. Prepare Buffer and set the ph 3. Mix Both

167 Tips on Method Validation. 67 Validation on Mobile Phases. The mobile phase shall be prepared number of times by following the documented SP and confirm if the same chromatograms can be obtained over and over. It is ideal to use a person who didn t develop the SP and make them prepare the same mobile phase to confirm if reproducible chromatograms can be obtained or not. It is also important to use a different instrument/system to confirm if the same chromatograms can be obtained or not as well.

168 Tips on Method Validation. 68 Validation on an Analytical Column. It is very important to keep a log/record on each column that you have in your laboratory. Columns that you are about to use could be contaminated or deteriorated by earlier methods that has been used in the past. In some cases, contaminated or deteriorated columns can give you better results coincidently.

169 Tips on Method Validation. 69 Validation on an Analytical Column. After optimizing your method, make sure to use a new column and confirm if you can obtain the same results/chromatograms. It is also ideal to use the same column with a DIFFERENT Lot/Batch number to confirm the same results/chromatograms can be obtained or not.

170 Different Lot/Batch Number of an Analytical column 70 Make sure the nd or 3 rd columns are packed with different lot/batch numbers.

171 GL Sciences Inc. HPLC. How to Select an HPLC Column What and Which column to use in case there is a separation problem 7

172 7 What if your First Choice Column didn t work out? Most samples could be analyzed without any problem if you follow the key points explained in the earlier presentation slides. However, in some cases, some samples may not be retained or separated as expected In such case, select an appropriate column depending on your purpose/goal.

173 What if your First Choice Column didn t work out?. If you prefer changing the elution pattern A) Change the Stereoselectivity Try an Ultra High Carbon Loading DS column... Inertsil DS-HL B) Use a π Electron Interaction Try a Phenyl type column.. InertSustain Phenylhexyl InertSustain Phenyl C) Use an Embedded Polar groups Interaction Try an Embedded type DS column... Inertsil DS-EP 73

174 74 What if your First Choice Column didn t work out?. If you prefer changing the elution pattern drastically Try a column that offers a different separation mode.. InertSustain Amide 3. If you prefer eluting all compounds more faster Try a Rapid Elution C8 column... InertSustainSwift C8 4. For Sugar Analysis Try a Amino column which prevents Anomer Reesolution and adsorption of Reducing Sugars. InertSustain NH

175 75 B) Use a π Electron Interaction InertSustain Phenylhexyl columns are bonded with phenylhexyl groups, which employs a phenyl ring with a hexyl (6-carbon) linker and is densely bonded to our newly developed ES silica gel delivering complementary selectivity to straight alkyl-chain columns, but with industry leading inertness, lot-to-lot reproducibility and low back pressure. Physical Properties Silica : Newly Developed Silica Gel (ES Silica) Particle Size : 3 μm, 5 μm Bonded Phase : Phenylhexyl Groups End-Capping : Complete Surface Area : 350 m /g Carbon Loading : 9 % Pore Size : 00 A Pore Volume : 0.85 ml/g USP Code : L ph Range : ph =.0~0.0 Hydrophobic Interaction Hydrophobic π-π Interaction

176 76 Different Selectivity to C8 Phase InertSustain C8 To Slightly Change Selectivity 3 4 5, Conditions Column Size Eluent Flow Rate Col. Temp. :40 Detection :UV 54 nm : 5 μm, mm I.D : A) CH 3 B) H A/B = 70/30, v/v :.0 ml/min Samples. Uracil. Caffeine 3. Phenol 4. Butylbenzene 5. o-terphenyl 6. Amylbenzene 7. Triphenylene To Dramatically Change Selectivity InertSustain Phenylhexyl To Achieve More Retention InertSustain Phenyl Polyaromatics are Eluted Later

177 77 Strong Retentivity InertSustain Phenylhexyl Inertsil Ph 6 InertSustain C8 Conditions Eluent : A) CH 3 B) H A/B = 70/30, v/v Flow Rate :.0 ml/min Col. Temp. :40 Detection :UV 54 nm Sample :. Uracil. Caffeine 3. Phenol 4. Butylbenzene 5. o-terphenyl 6. Amylbenzene 7. Triphenylene InertSustain Phenyl Inertsil C8-4 6 Inertsil Ph InertSustain Phenylhexyl show the strongest retentivity among the Phenyl phases available in the market.

178 78 Benefits of Highly Inert Packing Material Analysis of Strong Basic Compounds InertSustain Phenylhexyl Luna Phenyl-Hexyl H 3 C Conditions Eluent : A) CH 3 CN B) 5 mm K HP 4 (ph 7.0, KH P 4 ) A/B = 40/60, v/v :.0 ml / min Flow Rate Col. Temp. : 40 Detection : UV 30 nm Sample : Dextromethorphan H N CH3 H 3 C H N CH3 XBridge Phenyl Zorbax Eclipse Plus Phenyl-Hexyl

179 79 Benefits of Highly Inert Packing Material Analysis of Antidepressants InertSustain Phenylhexyl 3 Conditions Eluent Flow Rate Col. Temp. :40 Detection :UV 54 nm Sample : A) CH 3 CN B) 5 mm KH P 4 (ph 7.0, K HP 4 ) A/B = 50/50, v/v :.0 ml/min :. Imipramine hydrochloride. Amitriptyline hydrochloride 3. Clomipramine hydrochloride 4. Mianserin hydrochloride 4 N Luna Phenyl-Hexyl N Imipramine hydrochloride N Amitriptyline hydrochloride 4 3 Cl N H 3 C N H N N Clomipramine hydrochloride Mianserin hydrochloride

180 80 Benefits of Highly Inert Packing Material Analysis of Strong Basic Compound via LC-MS/MS 3.0e6 InertSustain Phenylhexyl Max..3e6 cps..0e6.0e6 Conditions System:GL QTRAP Column:InertSustain Phenylhexyl (3 μm, 50 x. mm I.D. ) InertSustain Phenyl (3 μm, 50 x. mm I.D. ) Eluent: A) CH 3 CN B) 0 mm CH 3 CNH 4 in H A/B = 30/70 (gradient mixer ), v/v Flow rate:00 μl/min Col. Temp.:40 Detection:SIM (ESI, Positive ) Injection Vol.: μl Sample:Dextromethorphan 50 ng/ml H 3 C e6.0e6.0e InertSustain Phenyl (Non End-capped column) Max..7e6 cps H N CH3 InertSustain Phenylhexyl employs a highly inert packing material which prevents secondary interaction between residual silanols delivering rapid results with higher sensitivity.

181 8 Benefits of Highly Inert Packing Material Analysis of Strong Acidic Compounds InertSustain Phenylhexyl Luna Phenyl-Hexyl 3 3 Conditions Eluent : A) CH 3 CN B) 0. % H 3 P 4 A/B = 5/75, v/v :.0 ml/min Flow rate Col. Temp. : 40 Detection : UV 54 nm Sample :. Brilliant Blue FCF. Phenol 3. Salicylic acid - 3 S S 3 - S N N + XBridge Phenyl Zorbax Eclipse Plus Phenyl-Hexyl. Brilliant Blue FCF 3 : Not Eluted

182 % Initial N 8 Extreme Durability Purging Conditions Column :4.6 mm I.D. x 50 mm, 5 μm Eluent :50 mm Triethylamine (ph 9.5) / CH 3 = 70/30, v/v Flow Rate :.0 ml/min Col. Temp. :50 Analytical Conditions Eluent :CH 3 CN/H = 65/35, v/v Flow Rate :.0 ml/min Col. Temp. :40 Detection :UV54 nm Sample :Naphthalene Minutes of Purging Some samples require high ph for dissolution or to maintain stability. InertSustainSwift C8 offers wide ph compatibility and high durability.

183 83 Applications InertSustain C8 Analysis of Analgesic Drugs Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) 5 mm KH P 4 (ph 3.0, H 3 P 4 ) A/B = 45/55, v/v Flow Rate :.0 ml/min Col. Temp. :40 Detection :UV 30 nm Sample :. Indomethacin. Ibuprofen Cl H 3 C N CH 3 H 3 C CH 3 H CH 3 Indomethacin Ibuprofen Change in Elution Pattern InertSustain Phenylhexyl

184 84 Applications Analysis of Vitamin B6 3 4 Conditions Column Size : 5 μm, mm I.D. Eluent : 0 mm Ammonium acetate Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 30 nm Sample :. Nicotinic acid. Pyridoxal 3. Pyridoxine 4. Nicotinamide N Nicotinic Acid N Pyridoxine N Pyridoxal NH N Nicotinamide

185 85 Applications Analysis of Capsaicin Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 B) H A/B = 60/40, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 80 nm Sample :. Capsaicin Impurity H H N

186 86 Applications Analysis of Antihistamine 3 Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) 5 mm KH P 4 (ph 7.0, K HP 4 ) A/B = 40/60, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 30 nm Sample :. Maleic acid. Chlorpheniramine 3. Triprolodine 4. Diphenhydramine Cl 4 N N Maleic Acid Chlorpheniramine N N N H 3 C Triprolodine Diphenhydramine

187 87 Applications Analysis of Antidepressants 3 4 Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) 5 mm KH P 4 (ph 7.0, K HP 4 ) A/B = 50/50, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm Sample :. Imipramine hydrochloride. Amitriptyline hydrochloride 3. Chlomipramine hydrochloride 4. Mianserin hydrochloride N N Imipramine hydrochloride N Amitriptyline hydrochloride Cl N H 3 C N H N N Chlomipramine hydrochloride Mianserin hydrochloride

188 88 Applications Analysis of Beta Blockers Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) 5 mm KH P 4 (ph 7.0, K HP 4 ) A/B = 0/80, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 0 nm Sample :. Atenolol. Pindolol 3. Acebutolol 4. Phenol 3 4 H N CH 3 HN H N CH 3 H N CH 3 CH Atenolol Pindolol CH 3 H N CH 3 H 3 C N H CH 3 Acebutolol

189 89 Applications Analysis of Calcium Antagonist Agents Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) 5 mm KH P 4 (ph 7.0, K HP 4 ) A/B = 40/60, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 35 nm Sample :.Nifedipine.Diltiazem CH H 3 C H 3 C H N CH 3 N CH 3 S N H CH 3 H H 3 C N CH3 Nifedipine Diltiazem

190 90 Applications Analysis of Indomethacin by European Pharmacopeia Indomethacin to be eluted around 8 minutes. Conditions Column : InertSustain Phenylhexyl ( 3 μm, mm I.D.) Eluent : A) 0 g/l CH 3 C in H B) CH 3 CN A/B = 70/30 min - 70/30 9 min - 50/50 min - 50/50-0 min - 70/30-9 min - 30/70 6 min - 30/70, v/v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 54 nm Injection Vol. : 0 μl Cl N H 3 C CH 3 Indomethacin

191 9 Applications Analysis of Etopocide by Chinese Pharmacopeia NLT: N > 3,000 Conditions Column : InertSustain Phenylhexyl (5 μm, mm I.D.) Eluent : A) CH 3 CN/33 mm CH 3 CNa (ph 4, CH 3 C) = 0/80 v,v B) CH 3 CN/33 mm CH 3 CNa (ph 4, CH 3 C) = 60/40 v,v A/B = 00/0-5 min - 00/0-5 min - 40/60-0 min - 40/60 - min - 0/00-3 min - 0/00 - min - 00/0-3 min - 00/0, v/v Flow Rate :.0 ml/min Col. Temp. : 0 Detection : UV 54 nm Injection Vol. : 5 μl H H 3 C H H H H H H H H H H 3 C CH 3

192 9 B) Use a Strong π Electron Interaction InertSustain Phenyl delivers an extremely unique reverse phase characteristics that are critical to resolving compounds that could not be separated on a C8 or C8 phase. InertSustain Phenyl provides not only pi-pi interactions, but also hydrogen bonding secondary interactions, which results in retaining polar compounds at the same time. Exceptional separation for metabolites. Physical Properties Silica : Newly Developed Silica Gel (ES Silica) Particle Size : 3 μm, 5 μm Bonded Phase : Phenyl Groups (Directly Bonded to the Silica) End-Capping : None Surface Area : 350 m /g Carbon Loading : 0 % Pore Size : 00 A Pore Volume : 0.85 ml/g USP Code : L ph Range : ph = ~7.5 Evolved Surface Si A Phenyl group is directly bonded to the silica-gel to maximize the effect of an electron affinity.

193 93 Difference between a Phenyl and Alkyl-Phenyl Column InertSustain Phenyl Evolved Surface Si Mainly pi-pi Interaction = Very Different Selectivity compared to an DS (C8) Alkyl-Phenyl type Hydrophobic Interaction is also available = Results in Similar Selectivity as an DS (C8) Butylphenyl Groups (C4-Ph)

194 94. Extreme Selectivity - Recognition Performance of pi-pi Interactions - Polyaromatic compounds have more pi-electrons compared to hydrocarbon compounds. InertSustain Phenyl offers strong retentivity for polyaromatic compounds such as Anthracene compared to Butylbenzene which is a hydrocarbon compound. As a result, InertSustain Phenyl delivers maximum selectivity compared to those commercially available Phenyl columns in the market. InertSustain Phenyl α(ant/bb) =.60 6 Analytical Conditions Eluent Flow Rate Col. Temp. Detection Sample :A) CH 3 B) H A / B = 60 / 40, v / v :.0 ml / min : 40 : UV 54 nm :.Uracil.Ethylbenzene 3.Naphthalene 4.Propylbenzene 5.Butylbenzene 6.Anthracene 5.Butylbenzene 6.Anthracene

195 95. ther Brand s Phenyl Columns - Recognition Performance of pi-pi Interactions - 3,4 6 5 α(ant/bb) = α(ant/bb) = α(ant/bb) = Spherisorb Phenyl Ascentis Phenyl XBridge Phenyl

196 96. Extreme Selectivity - Stereoselectivity Test - When conducting a selectivity test (Tanaka mixture test), the result on the separation between Alkylbenzene and Polyaromatic compounds indicates the feature of an HPLC column. InertSustain Phenyl has a feature of eluting the steric structured, o-terphenyl faster than the planar structured, Triphenylene. Although InertSustain Phenyl is a Phenyl column, it also offers high recognition performance for steric (non-planar) compounds.,3 InertSustain Phenyl Analytical Conditions Eluent Flow Rate Col. Temp. Detection Sample :A) CH 3 B) H A / B = 70 / 30, v / v : 0.8 ml / min : 40 : UV 54 nm :.Uracil. Caffeine 3. Phenol 4. Butylbenzene 5. o-terphenyl 6. Amylbenzen 7. Triphenylene 5. o-terphenyl 7. Triphenylene

197 97. ther Brand s Phenyl Columns - Stereoselectivity Test - 7,4 7 6,3 3 5, Spherisorb Phenyl Ascentis Phenyl XBridge Phenyl

198 98 Creating Significantly different Selectivity than an DS Column As shown above, InertSustain Phenyl elutes those alkylbenzenes and polyaromatics into two groups, whereas an DS column show less selectivity. InertSustain C8 InertSustain Phenyl 3 4 5, Analytical Conditions Eluent : A) CH 3 B) H A / B = 70 / 30, v / v Flow Rate :0.8 ml / min Col. Temp.:40 Detection :UV 54 nm Sample :. Uracil. Caffeine 3. Phenol 4. Butylbenzene 5. o-terphenyl 6. Amylbenzene 7. Triphenylene

199 Analysis of Estrogens Estrogen is a group of female hormones composed of estrone, estradiol and estriol that influences the functions of the female reproductive tract. It is hard to separate these compounds using an DS column, but InertSustain Phenyl delivers complete separation with less time. InertSustain C8, InertSustain Phenyl Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 B) H A / B = 60 / 40, v /v Flow Rate : 0.8 ml / min Col. Temp. : 40 Detection : UV 0 nm Sample : H H H. Estriol H CH 3 H CH 3 CH H H H H CH 3 H. Estradiol CH H H H H H H 3. Ethynylestradiol 4. Estrone 99

200 Analysis of Methylhippuric Acids It is again hard to separate m- (meta) and p- (para) in Methylhippuric Acid (a metalobile of Xylene) using an DS or other brand s Phenyl columns. InertSustain Phenyl provides separation of these isomers due to its high stereoselectivity with a popular mobile phase condition. InertSustain C InertSustain Phenyl 3 Inertsil Ph-3 Analytical Conditions Column Size : 5 μm, mm I.D. Eluent Flow Rate :.0 ml/min Col. Temp. :40 Detection :UV 30 nm Sample :,3 : A)CH 3 B)5 mm KH P 4 (ph = 3.0, 5mM H 3 P 4 ) A / B = 5 / 75 N H. o-methylhippuric acid N H. m-methylhippuric acid 3 N H 3. p-methylhippuric acid

201 0 Analysis of Non-Steroid Anti-Inflammatory Drugs It is hard to separate these type of drugs using conventional DS columns, but InertSustain Phenyl show superior separations with sharp peaks. InertSustain C8, Analytical Conditions Column Size : 5 μm, mm I.D. Eluent Flow Rate:0.8 ml / min Col. Temp.:40 Detection:UV 30 nm Sample : : A) CH 3 B) 5 mm KH P 4 (ph = 3.0, 5mM H 3 P 4 ) A / B = 60 / 40, v / v InertSustain Phenyl 3 H 3 C CH 3 H CH 3. Ibuprofen Cl Cl N NH CH 3 H 3 C Cl. Diclofenac Sodium 3. Indomethacin

202 Analysis of Catechins Catechins are known to inhibit cancers and reduce cholesterol levels in blood. As shown below, the elution pattern or selectivity of Epicatechin and Epigallocatechin gallate were different on InertSustain C8 and Phenyl. InertSustain C InertSustain Phenyl 5 6 Analytical Conditions Column Size: 5 μm, mm I.D. Eluent: A)CH 3 B)5 mm KH P 4 (ph = 3.0, 5 mm H 3 P 4 ) A / B = 0 /80, v / v Flow Rate :.0 ml/min Col. Temp. : 40 Detection : UV 80 nm H. Gallocatechin H. Catechin H 3. Epicatechin H H H Epigallocatechin gallate 5. Epicatechin gallate 6. Gallocatechin gallate 0

203 Analysis of Cardiac Glycosides When analyzing samples that would have a small influence to pi-pi interactions, the selectivity between an DS and Phenyl column will be similar in most cases. Digotoxin is a cardiac glycoside which has a high molecular weight and tends to elute very late. InertSustain Phenyl delivers the same selectivty as an DS column, but with rapid analysis. Analytical Conditions InertSustain C InertSustain Phenyl Digoxin 3. Digotoxin H H3 C Column Size Eluent 3 H H H H H3C : 5 μm, mm I.D. : A) CH 3 B) H A / B = 60 / 40, v / v Flow Rate :0.8 ml / min Col. Temp. :40 Detection :UV 0 nm Sample : H H H H H H3 C H. uabain 3 H H H 03

204 Stereoselectivity (Tri/o-ter) 04 Comparison of Selectivity between ther Brand s Phenyl columns InertSustain Phenyl InertSustain Phenyl High Selectivity Conditions Eluent : A) CH 3 B) H A/B = 70/30, v/v :.0 ml/min Flow Rate Col. Temp. :40 Detection :UV 54 nm Sample :. Uracil. Caffeine 3. Phenol 4. Butylbenzene 5. o-terphenyl 6. Amylbenzene 7. Triphenylene Selectivity Low Low InertSustain Phenylhexyl Strong Retentivity k(amylbenzene) Retentivity High InertSustain Phenyl Inertsil Ph Inertsil Ph-3 XBridge Phenyl Luna Phenyl-Hexyl Zorbax Eclipse Plus Phenyl-Hexyl InertSustain Phenylhexyl InertSustain Phenylhexyl show the strongest retentivity among the Phenyl phases available in the market. InertSustain Phenyl show the highest stereoselectivity.

205 05 Comparison Table Column Name Bonded Phase Structure Size (mm) Particle Size (μm) Surface Area (m /g) Pore Size (A ) Pore Volume (ml/g) Carbon Loading (%) InertSustain Phenyl Phenyl Groups (C0-Ph) Spherisorb Phenyl Phenyl Groups (C0-Ph) Ascentis Phenyl Butylphenyl Groups (C4-Ph) XBridge Phenyl Hexylphenyl Groups (C6-Ph) Luna Phenyl-Hexyl Hexylphenyl Groups (C6-Ph) * The Cn indicates the carbon number of the spacer (alkyl chain). Phenyl Groups (C0-Ph) Butylphenyl Groups (C4-Ph) Hexylphenyl Groups (C6-Ph)

206 Comparison of Selectivity between ther Brand s Phenyl columns Conventional Phenyl Columns Phenyl Groups Silica Gel InertSustain Phenyl The surface area is slightly smaller than a conventional Phenyl column, however, the bonding density of Phenyl groups are high. This results in delivering a very strong pi-pi interactions with superior stereoselectivity. Phenyl Groups Silica Gel 06 06

207 07 Explanation of Analytical Tests and Conditions Polycyclic Aromatic Test The resolution of Butylbenzene and Anthracene is determined to confirm the pi-pi interaction by the pi-electron. Stronger the pi-pi interactions, the more resolution between Butylbenzene and Anthracene. Selectivity Test Butylbenzene Anthracene The selectivity is confirmed by checking the separation between Bases and Acids and Alkylbenzene and Polycyclic Aromatic compounds. Analytical Conditions Eluent Flow Rate Col. Temp. Detection Injection Vol. Analytical Conditions Eluent Flow Rate Col. Temp. Detection Injection Vol. :A) CH 3 B) H A / B = 60 / 40, v / v :.0 ml / min :40 :UV 54 nm :5 μl :A) CH 3 B) H A / B = 70 / 30, v / v :0.8 ml / min :40 :UV 54 nm :5 μl Sample. Uracil. Ethylbenzene 3. Naphthalene 4. Propylbenzene 5. Butylbenzene 6. Anthracene Sample. Uracil. Caffeine 3. Phenol 4. Butylbenzene 5. Amylbenzene 6. o-terphenyl 7. Triphenylene Position Isomer Test 3 types of Cresols were injected to confirm the stereoselectivity of the column. CH 3 CH 3 CH 3 Analytical Conditions Eluent Flow Rate Col. Temp. Detection Injection Vol. :A) CH 3 B) H A / B = 0 / 80, v / v :0.8 ml / min :40 :UV 54 nm :5 μl Sample. o-cresol. m-cresol 3. p-cresol

208 08 Explanation of Analytical Tests and Conditions Structural Analog Test Hydrocortisone and Prednisolone has a similar molecular structure and is hard to separate using an DS column. These compounds are used to determine the strength of pi-pi interactions of the column and selectivity. No Double-bonding H H H H Hydrocortisone (Cortisol) Double-bonding H H H H Prednisolone Analytical Conditions Eluent : A) CH 3 B) H A / B = 40 / 60, v / v Flow Rate :0.8 ml/min Col. Temp. Detection :40 :UV 0 nm Sample. Hydrocortisone (Cortisol). Prednisolone Pyridine and Phenol Test Pyrdine is injected to confirm the inertness to basic compounds. Tailing can be confirmed when the packing material contains residual silanol groups. N Analytical Conditions Eluent : A) CH 3 CN B) H A / B = 30 / 70, v / v Flow Rate :0.8 ml/min Col. Temp. :40 Detection :UV 54 nm Sample. Uracil. Pyridine 3. Phenol Formic Acid and Acetic Acid Test Formic Acid and Acetic Acid are injected to confirm the inertness to acidic compounds. Tailing will occur when the surface of the packing material is basic. Analytical Conditions Eluent :0.% H 3 P 4 Flow Rate :0.8 ml/min Col. Temp. :40 Detection :UV 0 nm Sample. Formic acid. Acetic acid

209 09 Phenyl Columns Comparison Polycyclic Aromatic Test InertSustain Phenyl Ascentis Phenyl XBridge Phenyl ;Uracil ;Ethylbenzene 3;Naphthalene 4;Propylbenzene 5;Butylbenzene 6;Anthracene Spherisorb Phenyl Luna Phenyl-Hexyl Analytical Conditions 3, Eluent Flow Rate Col. Temp. Detection Injection Vol. :A) CH 3 B) H A / B = 60 / 40, v / v :.0 ml / min :40 :UV 54 nm :5 μl

210 Phenyl Columns Comparison Selectivity Test InertSustain Phenyl Ascentis Phenyl XBridge Phenyl ; Uracil ; Caffeine 3; Phenol 4; Butylbenzene 5; Amylbenzene 6; o-terphenyl 7; Triphenylene, 3 7, , Spherisorb Phenyl Luna Phenyl-Hexyl Analytical Conditions 7, , 7 Eluent Flow Rate Col. Temp. Detection Injection Vol. :A) CH 3 B) H A / B = 70 / 30, v / v :0.8 ml / min :40 :UV 54 nm :5 μl

211 Phenyl Columns Comparison Position Isomer Test InertSustain Phenyl ; o-cresol ; m-cresol 3; p-cresol 3 Ascentis Phenyl, 3 XBridge Phenyl, Spherisorb Phenyl Luna Phenyl-Hexyl Analytical Conditions,, 3, 3 Eluent Flow Rate Col. Temp. Detection Injection Vol. :A) CH 3 B) H A / B = 0 / 80, v / v :0.8 ml / min :40 :UV 54 nm :5 μl

212 Phenyl Columns Comparison Structural Analog Test InertSustain Phenyl. Cortisol. Prednisolone Ascentis Phenyl XBridge Phenyl, , Spherisorb Phenyl Luna Phenyl-Hexyl Analytical Conditions Eluent : A) CH 3 B) H A / B = 40 / 60, v / v Flow Rate :0.8 ml/min Col. Temp. :40 Detection :UV 0 nm

213 3 Phenyl Columns Comparison Pyridine and Phenol Test InertSustain Phenyl. Uracil. Pyridine 3. Phenol Ascentis Phenyl XBridge Phenyl Spherisorb Phenyl Luna Phenyl-Hexyl Analytical Conditions 3 3 Eluent : A) CH 3 CN B) H A / B = 30 / 70, v / v Flow Rate :0.8 ml/min Col. Temp. :40 Detection :UV 54 nm

214 4 Phenyl Columns Comparison Formic Acid and Acetic Acid Test InertSustain Phenyl. Formic acid. Acetic acid Ascentis Phenyl XBridge Phenyl Spherisorb Phenyl Luna Phenyl-Hexyl Analytical Conditions Eluent :0.% H 3 P 4 Flow Rate :0.8 ml/min Col. Temp. :40 Detection :UV 0 nm

215 5 GL Sciences Phenyl columns Column Name Bonded Phase Structure Size (mm) Particle Size (μm) Surface Area (m /g) Pore Size (A ) Pore Volume (ml/g) Carbon Loading (%) InertSustain Phenyl Phenyl Groups (C0-Ph) Inertsil Ph-3 Phenyl Groups (C0-Ph) Inertsil Ph Phenethyl Groups (C-Ph) * The Cn indicates the carbon number of the spacer (alkyl chain). Phenyl Groups (C0-Ph) Phenethyl Groups (C-Ph)

216 6 GL Sciences Phenyl Columns Comparison Polycyclic Aromatic Test InertSustain Phenyl Inertsil Ph-3 Inertsil Ph ;Uracil ;Ethylbenzene 3;Naphthalene 4;Propylbenzene 5;Butylbenzene 6;Anthracene

217 GL Sciences Phenyl Columns Comparison Selectivity Test InertSustain Phenyl Inertsil Ph-3 Inertsil Ph ; Uracil ; Caffeine 3; Phenol 4; Butylbenzene 5; Amylbenzene 6; o-terphenyl 7; Triphenylene 6, 7,

218 GL Sciences Phenyl Columns Comparison Position Isomer Test InertSustain Phenyl Inertsil Ph-3 Inertsil Ph ; o-cresol ; m-cresol 3; p-cresol 3 3,

219 Structural Analog Test InertSustain Phenyl Inertsil Ph-3 Inertsil Ph. Cortisol. Prednisolone GL Sciences Phenyl Columns Comparison,

220 Pyridine and Phenol Test InertSustain Phenyl Inertsil Ph-3 Inertsil Ph. Uracil. Pyridine 3. Phenol GL Sciences Phenyl Columns Comparison

221 GL Sciences Phenyl Columns Comparison Formic Acid and Acetic Acid Test InertSustain Phenyl Inertsil Ph-3 Inertsil Ph. Formic acid. Acetic acid Peak was not eluted

222 For Sugar Analysis InertSustain NH is an ultra performance sugar analysis column using a weak ion-exchanger, which can be used in a HILIC mode as well. The introduction of primary amine groups prevents anomer resolution at low temperature analysis offering easy analysis for sugar methods. In addition, the newly developed ES (Evolved Surface) silica delivers highly reproducible results with exceptional stability and durability that will maintain performance over the lifetime of the method. Physical Properties Silica : Newly Developed Silica Gel (ES Silica) Particle Size : 3 μm, 5 μm Bonded Phase : Aminopropyl Groups End-Capping : None Surface Area : 350 m /g Carbon Loading : 7.0 % Pore Size : 00 A Pore Volume : 0.85 ml/g USP Code : L8 ph Range : ph = ~7.5

223 3 Benefits of InertSustain NH Your First Choice Column for Sugar Applications. Delivers highly reproducible and stable retention time with exceptional durability. Extremely small reduction of retention time even purging the column with acidic eluent 3. Easy Sugar analysis preventing anomer resolution 4. Preventing the adsorption of reducing sugars

224 . Exceptional Durability After the columns were equilibrated, the columns were then purged with an eluent and confirmed the reduction rate of retention time of Maltose InertSustain NH 00 Inertsil NH Reduction Rate of Retention Time of Maltose (%) Volume of Eluent Purged through the Column (ml) As shown above, InertSustain NH stabilizes with less time and provides reproducible retention time Luna NH Asahipak NH Unisil Q NH Unison UK-Amino Analytical Conditions Eluent : A)CH 3 CN B)H A / B = 75 / 5, v / v Flow Rate :.0 ml/min Col. Temp. :40 Detection :RI Sample :. Fructose. Glucose 3. Sucrose 4. Maltose 4

225 5. Small Reduction of Retentivity even Purging the column with Acidic Eluent Retention time and Peak shapes were monitored after purging the column with acidic eluent. InertSustain NH Inertsil NH Unisil Q NH st Injection After purging with acidic eluent Weaker Retention + Change in Peak Shapes Weaker Retention + Change in Peak Shapes As shown above, InertSustain NH delivers no fluctuation of Retentivity Caution! Fluctuation of retentivity will not be observed under neutral eluent conditions. In such conditions, sugars can be analyzed without any problem.

226 Column Cleaning/Flushing Procedure Caution! Polymer Base (ther brand s columns), Ex: Asahipak NHP-50 In General: Clean/Flush it with strong basic solvents When analyzing neutral sugars under acidic eluent conditions or Analyze neutral sugars with polymer columns flushed with acidic eluents Fluctuation in Retention time or degraded peak shapes are often observed. Asahipak NHP-50 4E Initial Chromatogram After purging with acidic eluent To prevent the above phenomenon, Sodium Hydroxide is used to clean/flush the column InertSustain NH Recommended Solvent: Clean/Flush it with Ammonium Formate or Ammonium Acetate Adsorption of reducing sugars will not be observed even flushing it with acidic eluents on InertSustain NH. * Please note that Ammonium Formate cannot be used when cleaning/flushing Inertsil NH. 6

227 7 3. Easy Sugar Analysis with N Anomer Resolution So, what is an Anomer Resolution??? After reduction of the sugar and saccharide, the terminal groups may have various types of cyclic forms in addition to the open-chain form. These saccharides exist as two diastereomers since the carbon atom of the terminal reducing group is asymmetric; these diastereomers are called anomers (alpha- and beta-anomers). Under certain conditions where the rate of conversion of such diastereomers is low, alpha-anomer and beta-anomer are separated as they pass through the column. This causes splitting or broadening of the peak as shown in the chromatogram below. CH 4 6 CH 5 3 CH CH Reducing Character H C H C 3 H C H 4 H C 5 H C 6 CH Not a Primary Amine Bonded Column Anomer Separation α-d-glucose Anomer β-d-glucose What about InertSustain NH??? InertSustain NH is a Primary Amine Bonded column and the column interior shows an alkaline condition, which results in preventing anomer resolution with easy sugar analysis. InertSustain NH

228 8 4. Preventing the Adsorption of Reducing Sugars Glucose and Maltose are known to be hard-to-elute reducing sugars. Commercially available NH columns often show adsorption of peaks due to the residual silanols in the packing material. Adsorption of reducing sugars will not be observed on InertSustain NH due to the introduction ES (Evolved Surface) silica, which has less silanols in the packing. InertSustain NH Asahipak NHP-50 Unison UK-Amino Unisil Q NH Polymer Type Column Analytical Conditions Eluent : A)CH 3 CN Sample :. Fructose ( mg/ml) B)H. Glucose (4 mg/ml) A / B = 75 / 5,v / v 3. Sucrose ( mg/ml) Flow Rate :.0 ml/min 4. Maltose (50 mg/ml) Col. Temp. : 40 Detection : RI CH Fructose CH Glucose CH CH Sucrose CH CH CH Maltose

229 Analysis of Potassium Guaiacolsulfonates Potassium Guaiacosulfonates are used as expectorants and often contained in cold medicines. InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 B) 50 mm KH P4 A / B = / 0, v / v Flow Rate :0.95 ml / min Col. Temp. :30 Detection :UV 79 nm Sample :. Guaiacol. Guaiacolsulfonate H CH 3 H S CH 3 K. Guaiacol. Guaiacolsulfonate

230 Analysis of Maltooligosaccharides InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) H A / B = 60 / 40, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :RI Sample :. Maltose (n=0). Maltotriose (n=) 3. Maltotetraose (n=) 4. Maltopentaose (n=3) 5. Maltohexaose (n=4) 6. Maltoheptaose (n=5) 3 CH CH CH n

231 Analysis of Ascorbic Acids Homocysteine reduction method is employed in the Japanese Food Sanitation Inspection Guidline, Food Additive Test. InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) 0 mm NaH P 4 C) 0.03 % Homocysteine D) CH 3 A / B / C / D = 60 / 0 /3 / 3, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 70 nm Sample :. Erythorbic acid. L-Ascorbic acid H H H H H H H H. Erythorbic Acid. Ascorbic Acid

232 Analysis of Glucosamine InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) H A / B = 75 / 5, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :RI Sample :. Xylitol. Glucosamine H H NH. Glucosamine

233 Analysis of Tocopherols (Vitamin E) InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) Ethyl acetate B) Hexane A / B = 30 / 70, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 90 nm R 3 Sample :. α-tocopherol R,R,R3;CH 3. β-tocopherol R,R3;CH 3 R;H 3. γ-tocopherol R,R3;CH 3 R;H 4. δ-tocopherol R3;CH 3 R,R;H R R

234 Analysis of Tetrasaccharides Acorbose, a tetrasaccharide is often used for controlling the blood sugar level for diabetes. InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) H A / B = 70 / 30, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :RI Sample :. Acarbose. Stachyose CH CH CH NH CH 3 CH CH CH CH CH. Acarbose. Stachyose

235 Analysis of Acesulfame-K Acesulfame potassium, a synthetic sweetener was analyzed under a condition based on the Japanese Food Sanitation testing method. InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) %H 3 P 4 A / B = 60 / 40, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 30 nm Sample :. Saccharin. Acesulfame potassium S NH S N K. Saccharin. Acesulfame potassium

236 Analysis of Allantoin Allantoin is a compound often used in hand cream and eye-drop. InertSustain NH Analytical Conditions Column Size : 5 μm, mm I.D. Eluent : A) CH 3 CN B) H A / B = 85 / 5, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :UV 0 nm Sample :. Allantoin H N N H N H NH. Allantoin

237 37 Comparison Table Column Name Bonded Phase Structure Size (mm) Particle Size (μm) Surface Area (m /g) Pore Size (A ) Pore Volume (ml/g) Carbon Loading (%) InertSustain NH Aminopropyl Groups Luna NH Aminopropyl Groups Asahipak NHP-50 Amino Groups (Polymer Base) Unison UK-Amino Aminopropyl Groups

238 38 Explanation of Analytical Tests and Conditions Sugar Test Peak shapes and retentivity were confirmed by using sugars such as Monosaccharides and Disaccharides. CH Fructose CH Sucrose CH CH CH CH Glucose Maltose CH Analytical Conditions Eluent : A)CH 3 CN B)H A / B = 75 / 5, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :RI Sample :. Fructose. Glucose 3. Sucrose 4. Maltose If fructose is in water, it would generally change the structure into either β-fructopyranose and β-fructofranose. Generally, the presence of β-fructopyranose will be more and this is another reducing sugar. CH β- Fructopyranose Cyclodextrin Test Columns bonded with secondary or tertiary amine groups may show poor separation between β-cyclodextrin and γ-cyclodextrin. n H H H H H H H H Analytical Conditions Eluent : A)CH 3 CN B)H A / B = 60 / 40, v / v Flow Rate :.0 ml / min Col. Temp. :40 Detection :RI Sample :. α-cyclodextrin (n=6). β-cyclodextrin (n=7) 3. γ-cyclodextrin (n=8)

239 Explanation of Analytical Tests and Conditions Sugar Alcohol Test Selectivity and retentivity were confirmed by using Sugar Alcohols. H H Erythritol Xylitol H H Analytical Conditions Eluent : A)CH 3 CN B)H A / B = 85 / 5 Flow Rate :.0 ml / min Col. Temp. :40 Detection :RI Sample :. Erythritol. Xylitol 3. Sorbitol 4. Mannitol Sorbitol Mannitol Nucleic Acid Test The retentivity in HILIC mode was confirmed by using Nucleic Acids. N H NH N H NH NH Thymine Uracil Adenine NH N N N N N H N N Analytical Conditions Eluent : A)CH 3 CN B)0mM-HCNH 4 A / B = 85 / 5 Flow rate :.0 ml / min Col. Temp. :40 Detection :UV 54 nm Sample :. Cytosine. Uracil 3. Guanine 4. Thymine 5. Adenine N H Cytosine N H N Guanine NH 39

240 40 NH Columns Comparison Sugar Test (Retentivity of Sugars) InertSustain NH ; Fructose ; Glucose 3; Sucrose 4; Maltose Asahipak NHP-50 Luna NH 00A Unison UK-Amino Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars. The retentivity is strong, but the elution order is different for Nucleic Acids as it is a polymer-base column. The column may be bonded with secondary or tertiary amine groups as it shows poor separation between β- Cyclodextrin and γ- Cyclodextrin and strong retention for Nucleic Acids. The retentivity is very weak and show adsorption to reducing sugars.

241 4 NH Columns Comparison Cyclodextrin Test InertSustain NH Asahipak NHP-50 Luna NH 00A Unison UK-Amino ; α-cyclodextrin ; β-cyclodextrin 3; γ-cyclodextrin Poor Resolution Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars. The retentivity is strong, but the elution order is different for Nucleic Acids as it is a polymer-base column. The column may be bonded with secondary or tertiary amine groups as it shows poor separation between β- Cyclodextrin and γ- Cyclodextrin and strong retention for Nucleic Acids. The retentivity is very weak and show adsorption to reducing sugars.

242 4 NH Columns Comparison Sugar Alcohol Test InertSustain NH Asahipak NHP-50 Luna NH 00A Unison UK-Amino ; Erythritol ; Xylitol 3; Sorbitol 4; Mannitol Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars. The retentivity is strong, but the elution order is different for Nucleic Acids as it is a polymer-base column. The column may be bonded with secondary or tertiary amine groups as it shows poor separation between β- Cyclodextrin and γ- Cyclodextrin and strong retention for Nucleic Acids. The retentivity is very weak and show adsorption to reducing sugars.

243 43 NH Columns Comparison Nucleic Acid Test InertSustain NH ; Thymine ; Uracil 3; Adenine 4; Cytosine 5; Guanine Asahipak NHP-50 Luna NH 00A Unison UK-Amino Change in Elution Pattern Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars. The retentivity is strong, but the elution order is different for Nucleic Acids as it is a polymer-base column. The column may be bonded with secondary or tertiary amine groups as it shows poor separation between β- Cyclodextrin and γ- Cyclodextrin and strong retention for Nucleic Acids. The retentivity is very weak and show adsorption to reducing sugars.

244 44 GL Sciences NH Columns Comparison Sugar Test (Retentivity of Sugars) InertSustain NH ; Fructose ; Glucose 3; Sucrose 4; Maltose Inertsil NH Unisil Q NH Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars. The retentivity is strong, but show adsorption to reducing sugars. Also, separation for sugar alcohols were not enough. The retentivity is strong, but show severe adsorption to reducing sugars.

245 45 GL Sciences NH Columns Comparison Cyclodextrin Test InertSustain NH ; α-cyclodextrin ; β-cyclodextrin 3; γ-cyclodextrin Inertsil NH Unisil Q NH Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars The retentivity is strong, but show adsorption to reducing sugars. Also, separation for sugar alcohols were not enough The retentivity is strong, but show severe adsorption to reducing sugars.

246 46 GL Sciences NH Columns Comparison Sugar Alcohol Test InertSustain NH ; Erythritol ; Xylitol 3; Sorbitol 4; Mannitol Inertsil NH Unisil Q NH Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars. The retentivity is strong, but show adsorption to reducing sugars. Also, separation for sugar alcohols were not enough. The retentivity is strong, but show severe adsorption to reducing sugars.

247 47 GL Sciences NH Columns Comparison Nucleic Acid Test InertSustain NH ; Thymine ; Uracil 3; Adenine 4; Cytosine 5; Guanine Inertsil NH Unisil Q NH Among those silica-based NH columns, InertSustain NH delivers the strongest retentivity. Also has no adsorption to reducing sugars. The retentivity is strong, but show adsorption to reducing sugars. Also, separation for sugar alcohols were not enough. The retentivity is strong, but show severe adsorption to reducing sugars.

248 Comparison of Retentivity between various HILIC Columns Inertsil Amide Inertsil NH InertSustain NH Analytical Conditions Eluent :A) CH 3 CN B) 0mM HCNH 4 A / B = 85 / 5, v / v Flow Rate :.0mL / min Col. Temp. :40 Detection :UV 54nm Sample :. Thymine. Uracil 3. Adenine 4. Cytosine 5. Guanine N N H N H NH N NH N N H NH N H NH N Retentivity N N H N Guanine is used to confirm the retentivity of each column in this experiment. N NH Inertsil Amide Inertsil NH > InertSustain NH > Inertsil HILIC 48

249 49 Sugars - Difference between Inertsil Amide - Addition of amines to the mobile phase or increase in column temperature are required when analyzing sugars using Inertsil Amide (Bonded Phase: Carbamoyl Groups) to prevent anomer resolution. However, the above mentioned procedures are not required when using InertSustain NH or Inertsil NH since it does not show any anomer resolution InertSustain NH 3 4 ( ( ( Inertsil Amide Analytical Conditions Eluent : A)CH 3 CN B)H A / B = 75 / 5 Flow Rate :.0 ml/min Col. Temp. :40 Detection :RI Sample :. Fructose (mg/ml). Glucose (4mg/mL) 3. Sucrose (mg/ml) 4. Maltose (50mg/mL) CH CH CH CH CH CH CH Fructose Glucose Sucrose Maltose Addition of TEA to the mobile phase or increase in column temperature are required when analyzing sugars using Inertsil Amide to prevent anomer resolution.

250 50 To Maximize Throughput There are approaches to shorten the analysis time Shorten the column length Increase the flow rate However, when using a conventional column The separation performance decreases when shortening the column length The operating back pressure increases when increasing the flow rate The separation is poor when the flow rate is not set at the optimum flow rate

251 5 HETP* (μm) How to Maximize Throughput using Sub- micron columns van Deemter-plot When using Sub- micron columns um 3um 4um 5um. In most cases, the separation performance increases when the flow rate is the same.. The optimum flow rate becomes wider and higher. Not only the analysis time can be shorter, but better separation performance can be obtained when using sub- micron columns by setting an optimum flow rate Linear Velocity (mm/s) 移動相の線速度 (mm/s) (Evaluated using Acetonitrile/Water = 50/50, Length 50 mm) * HETP: Height equivalent to a theoretical plate. A carryover from distillation theory: a measure of a column's efficiency. 5

252 5 What is Linear Velocity? Liner Velocity (mm/sec) = Flow Rate Column Cross-Section Area Linear velocity is the speed at which the solvent front travels the length of the column, which is calculated by dividing the column length by the retention time (t0) of an unretained component. Column I.D. Cross-Section Area Column Cross-Section Ratio Flow Rate 4.6 mm 6.6 mm 0.0 ml/min 3.0 mm 7. mm ml/min Equivalent Linear Velocity. mm 3.5 mm 0. ml/min mm/sec

253 Pressure 圧力 (MPa) (Mpa) 53 How to Maximize Throughput using Sub- micron columns The operating back pressure of Sub- micron columns can go up very easilty. The column lifetime can be shorter on sub- micron columns when comparing to those columns packed with bigger particle sizes above 3 um um 3um 4um 5um 移動相の線速度 Linear Velocity (mm/sec) (mm/s) Relationship between Linear Velocity and Back Pressure (Evaluated using Acetonitrile/Water = 65/35, Length 50 mm)

254 54 Applications of Sub- Micron Columns. mm I.D. 50 mm 3 µm 0. ml/min Neurotransmitter. Norepinephrine (NE). 3,4- Dihydroxyphenyl Acetate(DPAC) 3. 3,4- Dihydroxybenzylamine Hydrochlorid(DHBA) Hydroxyindoleacetic acid(5-hiaa) 5. Dopamine Hydrochloride(DA) 6. Homovanillic acid (HVA) 7. Serotonin Hydrochloride(5-HT). mm I.D. 50 mm µm 0.6 ml/min Reducing the analysis time by /9 System : LC800 system (ECD Detector) Column : Inertsil DS-3 Flow Rate : 0. ml/min or 0.6 ml/min Eluent : A) Methanol B) 6 mm Citric Acid, 8 mm Dibasic Potassium Phosphate, 40 mg/l SS, 50.8 mg/l EDTA Na A/B = 5/85(v/v) (gradient mixer) Column Temp. :35 Detection : ECD 00 mv response.0 s Injection Vol. : 5 μl ( ppm each)

255 55 Applications of Sub- Micron Columns 50 mm length column is the most popular length when using sub- micron columns. However, sometimes the desired efficiency could not be obtained. Analysis time Reduced by /6 3.0 x 50 mm, 5 um, 0.4 ml/min, 6. MPa x 50 mm, um,.0 ml/min, 3. MPa Analytical Conditions Column : Inertsil DS-3 Eluent : A: CH 3 CN B: 0. % H 3 P 4 A/B = 5/85 Col. Temp. : 40 Detector : PDA 75 nm Aromatic organic acid. Gallic acid (0.7 mg/ml). Vanillic acid (0.7 mg/ml) Vanillin (Impurity derived from ) 3. p-coumaric acid (0.7 mg/ml) 4. Ferulic acid (0.7 mg/ml) 5. m-coumaric acid (0.7 mg/ml)

256 56 Long Column Length 3 μm columns can be Effective Inertsil DS-3 (μm,. x 50mm), Flow Rate 500μL/min, Back Pressure = 6.4MPa Basic drugs. Ranitidine. Scopolamine 3. Naltrexone 4. Acetaminophen 5. Theophyline 6. Metoprolol 7. Caffeine 8. Chlorpheniramine 9. Propranolol 0. iphenhydramine. Doxepin. Amitriptyline 3. Reserpine 4. Isopropylantipyrine 5. Ketoprofen 6. Warfarin 7. Capsaicin 8. Dihydrocapsaicin Inertsil DS-3 HP (3μm,. x 75mm), Flow Rate 750μL/min, Back Pressure = 8.9MPa Less influence of diffusion by selecting a longer column length Analytical Conditions Column : Inertsil DS-3 or Inertsil DS-3 HP Eluent : A) CH 3 CN B) 0.05% HC in H A/B = 5/95 5 min - 95/5, v/v Col. Temp. : 40 Detection : LC/MS/MS (4000Q TRAP TM : ESI, Positive, MRM) Injection Vol. : 0 μl * The above back pressures were recorded at the initial eluent condition.

257 57 Rapid Analysis with 3 um Columns Analysis of DNPH Aldehydes InertSustain C8 (Maximum perating Back Pressure: 0 MPa) Flow Rate: 0.5 ml/min Back Pressure: 6.0 MPa Analytical Conditions Column : InertSustain C8 or InertSustain C8 HP (3μm, 50 x 3.0 mm I.D.) Eluent :CH 3 CN/H /THF = 35/55/0, v/v/v Col. Temp. : 40 Detection : UV 360 nm Rapid analysis without decreasing the separation efficiency.formaldehyde.acetaldehyde 3.Acetone 4.Acrolein 5.Propionaldehyde 6.Crotonaldehyde 7.Methylethylketone InertSustain C8 HP.5 ml/min 47. MPa 8.Methacrolein 9.n-Butyraldehyde 0.Benzaldehyde.n-Valeraldehyde.m-Tolualdehyde 3.Hexanal (DNPH Deriv.) (Maximum perating Back Pressure: 50 MPa) mvolt min mvolt minutes faster min

258 Volts Rapid Analysis Maintaining the Separation Efficiency using 3 um Columns 50 mm x 3.0 mm I.D. 3 µm 0.4 ml/min 7.5 MPa 3.8min. Insufficient Separation Time 30 (min) 50 mm x 3.0 mm I.D. 3 µm 0.4 ml/min.0 MPa Volts Increasing the column length 3.min. Separation achieved, but increase in analysis time Time 0.8 ml/min 5.0 MPa Volts Increasing the flow rate.min. 50 mm x 3.0 mm I.D 3 µm 0 Separation achieved with shorter analysis time without sacrificing the separation efficiency 0 0 Time 30 (min) 30 (min) 58

259 59 Summary on the Benefits of HP type 3 um Columns Less column clogging issues compared to sub -micron columns. Can be used on a standard HPLC system. Generates less column back pressure compared to sub -micron columns.

260 60 Summary of Maximizing Throughput using an Appropriate Column Usage of Sub- micron columns can reduce the analysis time. However, the operating back pressure would be very high. 3 um columns can be used as well as long as the column s withstanding operating back pressure allows. Usage of long column length can be effective when requiring to maintain the separation efficiency. To remove impurities that elutes after your target samples, a column switching method is an effective way to obtain ideal chromatograms.

261 GL Sciences Inc. HPLC GL Sciences Rigorous Quality Control Program 6

262 6 Benefits of GL Sciences HPLC Columns Features High Purity, Spherical Silica-gel Highly Uniformed Particle Size Distribution Highly Stable Chemical Bonding Extreme Endcapping Treatment Rigorous Inspection Tests (More than 0 Tests) Benefits High Theoretical Plates Low Back Pressure High Durability Lower Adsorption Batch-to-Batch Reproducibility

263 63 Benefits of GL Sciences HPLC Columns The Biggest Feature is We synthesize the material of the silica gel and conduct chemical bonding, inspection and the packing of the column by ourselves. Highly Stable Results Excellent After-Sales Support The Process Continues to Evolve for further Customer Satisfaction

264 Highly Pure Base Silica-gel Completely Spherical Smooth Surface Synthesized from Pure Ingredients Focused only for Chromatography The Entire Manufacturing Process is done within GL Sciences Inc. in Japan Inertsil Silica Gel ther Brand s Silica Gel 64

265 Highly Pure Base Silica-gel 65

266 Highly Pure Base Silica-gel 66

267 67 Influences of Metal Impurities Metal Impurities (ppm)

268 GL Sciences Rigorous Quality Control Program Scanning Electron Microscope Sphericity and surface smoothness of Silica-gel Atomic Emission and Inductivity Coupled Plasma Trace Metals Nitrogen Adsorption & Laser Ray Particle Analyzer Particle Size and its Uniformity Surface Area Pore Diameter and Pore Volume 9 Si-NMR Residual Silanol Chromatographic Tests Inertness Durability Selectivity (Bases, Acids, Complexes) (Low ph, High ph) (Hydrophobicity, Stereoselectivity, Hydrogen-Bonding Capacity) 68

269 69 GL Sciences Rigorous Quality Control Program Evaluation Test of Inertness to Basic compounds from Lot-to-Lot. Pyridine. Phenol LT# VQ5-43 LT# VQ5-44 LT# VQ5-45

270 70 GL Sciences Rigorous Quality Control Program Evaluation Test of Inertness to Acidic compounds from Lot-to-Lot. Formic Acid. Acetic Acid LT# VQ5-43 LT# VQ5-44 LT# VQ5-45

271 7 GL Sciences Rigorous Quality Control Program Evaluation Test of Inertness to Chelating compounds from Lot-to-Lot. xine-copper. Caffeine LT# VQ5-43 LT# VQ5-44 LT# VQ5-45

272 7 GL Sciences Rigorous Quality Control Program Evaluation Test of Selectivity k (n-amylbenzene) / k (n-butylbenzene) : Hydrophobicity of an DS k (Triphenylene) / k (o-terphenyl) : Stereoselectivity of an DS k (Caffeine) / k (Phenol) : Hydrogen-bonding capacity of an DS 7 ) Uracil ) Caffeine 3) Phenol ) Butylbenzne 5) o-terphenyl 6) Amylbenzene 7) Triphenylene 0 0 0

273 73 GL Sciences Rigorous Quality Control Program Evaluation Test of Selectivity from Lot-to-Lot Column : Inertsil DS-3V 50 x 4.6 mmi.d. Eluent : CH 3 / H = 80 / 0 Detector : UV 54nm Col.Temp.: 40 A:Hydrophobicity of the DS B:Stereoselectivity of the DS C:Hydrogen-bonding capacity of the DS A=.47 B=.3 C=0.49 A=.47 B=.30 C=0.5 A=.47 B=.30 C=0.5 LT# VQ5-68 LT# VQ5-03 LT# VQ5-06

274 74 GL Sciences Rigorous Quality Control Program Evaluation Test of Residual Silanol by 9 Si-NMR. Isolated silanol Si- Geminal silanol Si-() Silica gel Si-(Si) 4 --Si-(CH 3 ) 3 Bonding of an DS (C8) group --Si-C 8 H 37 Residual silanol End-capping

275 75 GL Sciences Rigorous Quality Control Program Evaluation Test of Residual Silanol by 9 Si-NMR. LT# VQ5-68 LT# VQ5-03 LT# VQ5-06

276 GL Sciences Rigorous Quality Control Program 9 Si-NMR Spectrum of Commercially Available DS Columns Residual silanol Residual silanol Brand A Brand B Residual silanol Residual silanol Brand C Brand D 76

277 GL Sciences Inc. HPLC H P L C Troubleshooting 77

278 78 Troubleshooting : Excessively High System Back Pressure The following three patterns can be considered The system back pressure is relatively higher than usual The system back pressure is relatively lower than usual The system back pressure is fluctuating or unstable

279 When the System Back Pressure is relatively Higher than usual Find out where the high pressure is generating Disconnect the column and connect an union Disconnect one by one the segments of tubing downstream * ther possible causes : Fluctuation of column oven temperature or the flow of the pump, which are generated from irreproducible analytical conditions. 79

280 80 When the System Back Pressure is relatively Higher than usual If the source of the problem/cause is determined. * When the column is deteriorated or the column inlet filter is clogged If the guard column is deteriorated Replace it with a new guard column If the analytical column is deteriorated Wash the column Refer to the washing method for reversed phase If the column inlet filter is clogged Wash the column (or replace the inlet filter) * When detector cell or tubing is clogged If the cell is clogged Wash the cell Wrap the cell with adsorbent cottons and place it inside an ultrasonic bath and wash it with water twice and methanol once. If the tubing is clogged Replace it with a new tubing *3 When the tubing is damaged due to over tightened fittings (Ex: Teflon tubing) Cut the tubing just short of the damaged tubing and then install a new fitting and tighten it *4 When the injector tubing, valve or needle is clogged Either wash it or replace it with a new part Periodically replace consumable parts such as rotor seals by referring to the manufacturer s replacement guidelines or instructions. *5 When the pump tubing or line filter is clogged Either wash it or replace it with a new part Periodically replace consumable parts such as line filters by referring to the manufacturer s replacement guidelines or instructions.

281 8 General Method of Washing a Reversed Phase Column 00% Prewash Wash 3Equilibration Concentration of rganic Solvent 5% 0% Washing Time Eluent composition used in the analysis Prewash Wash 3Equilibration :Introduce water at first to remove salts when the eluent contains salts. Then introduce an organic solvent. :In general, use Acetonitrile or Methanol. :After washing the column, equilibrate the column with the eluent used in the analysis. In case the eluent contains salts, equilibrate with water first. Then introduce an organic solvent and finally introduce the eluent composition used in the analysis.

282 8 Example of Washing a Reversed Phase Column The organic solvent can be either Acetonitrile or Methanol Reversed Phase General Analysis Reversed Phase Ion-Pair Reversed Phase Protein, Peptides 5% 5% rganic 有機溶媒 Solvent 5-30min 5% 5% rganic 有機溶媒 Solvent 30-0min 5-30min A:0.%TFA 5% B: 有機溶媒 0.%TFA in CH 3 CN A 00% B 00%, 5-30min Gradient x 3 times 00% rganic Solvent 5-60min 00% rganic Solvent 5-60min 00% rganic Solvent 5-60min 5-50% rganic Solvent 5min Storage 5-50% rganic Solvent 5min Storage 5-50% rganic Solvent 5min Storage Equilibrate it with the eluent Remarks Equilibrate it with the eluent Equilibrate it with the eluent When the eluent contains salts, always introduce purified water initially to prevent precipitation of salts in the column. When analyzing proteins, always introduce purified water initially to prevent proteins being denatured in the pore of the silica gel. In case Acetonitrile/Methanol cannot wash out the contaminants, try using IPA (Isopropyl alcohol). When washing the column, set the flow rate from 0.5 to.0 ml/min.

283 83 When the System Back Pressure is relatively Higher than usual Frequently Encountered Problems: The system back pressure starts to increase after 0 injections. The cause was on the analytical column. The analytical column was Inertsil DS-3 3um. mmi.d. x 50mm Approach on Solving the issue Wash the column. Use a Guard column. 3 Use a bigger particle size packing material. (Ex: Inertsil DS-3 4um) 4 Reconsider the sample preparation method.

284 84 Protect your Analytical Column Recommendation of using a Guard Column Purpose: To retain impurities/contaminants and to protect the analytical column. Removal of highly fat-soluble impurities in sample (Reversed Phase) Removal of highly polar impurities in sample (Normal Phase) Removal of impurities/contaminants in mobile phase or sample. Removal of impurities/contaminants from the pump. The actual picture image of an analytical column Installation of a Guard Column Install the guard column front of the analytical column. Try to make the tubing as short as possible. when using a guard column The actual picture image of an analytical column without using a guard column Guard Column Injector Analytical Column Detector

285 85 When the System Back Pressure is relatively Lower than usual Find out where the cause is coming from. Make sure there is no leak From the joint ver tighten the joint only once. If this doesn t work, replace to a new joint or tubing. Lower part of the pump head Replace the plunger seal. (Refer to the instruction manual). Make sure there are no air bubbles generating in the tubings Insufficient degassing Degas the solvent more. (Make sure the Degasser is turned N) Clogging of solvent filter Either clean the solvent filter or replace to a new one. 3. Differentiation of Column ven Temperature Make sure the set column oven temperature and the actual oven temperature is matching. If not, make necessary adjustments by referring to the instruction manual. 4. Make sure the pressure sensor of the pump is functioning normally Make sure the zero point is set correctly by referring to the instruction manual.

286 86 When the System Back Pressure is Fluctuating or Unstable Find out where the cause is coming from. Make sure the pump functioning correctly Contaminated check valve of the pump Either wash or replace it with a new one by referring to the instruction manual.. Make sure there are no air bubbles generating in the tubings Insufficient degassing Degas the solvent more. (Make sure the Degasser is turned N) Clogging of solvent filter Either clean the solvent filter or replace to a new one.

287 87 Troubleshooting : When the Peak Shape is Deteriorated The following two patterns can be considered The Actual Sample and Eluent Reconfirm the sample diluent Reconfirm the injection volume Reconfirm the buffer used in the eluent The Instrument Reconfirm the tubing and cell volume Reconfirm the flow of the pump Reconfirm whether the detector signal is saturated or not Reconfirm whether the column is not being overloaded or not Reconfirm whether the column is deteriorated or not

288 88 Sample Diluent The effect of Sample Diluent Pesticides. Methamidophos 0 mg/l. Acephate 0 mg/l The sample was dissolved using Analytical Condition: Column : Inertsil DS-SP (5 μm, 50 x 4.6 mm I.D.) Eluent : CH 3 CN / H = 5 / 95 (gradient mixer) Flow Rate :.0 ml/min Col.Temp. : 40 Detection : UV 5 nm Injection Vol. : 50 μl 00% Acetonitrile 50% Acetonitrile/Purified Water Purified Water only

289 89 Buffer. Benzoic Acid 00 mg/l. Benzyl Alcohol 00 mg/l C Analytical Condition: Column : Inertsil DS-3 (4 μm, 50 x 3.0 mm I.D.) Eluent : CH 3 CN / 0 mm Buffer (ph 4.5) = 5 / 85 (gradient mixer) Flow Rate : 0.4 ml/min Col.Temp. : 40 Detection : UV 5 nm Injection Vol : 5 μl 0 mm Acetic Acid Buffer (ph 4.5): Acetic Acid or Sodium Acetate solutions were prepared to 0 mm each and adjusted the ph at mm Phosphoric Acid Buffer (ph 4.5): Phosphoric Acid or Sodium Dihydrogen Phosphate were prepared to 0 mm each and adjusted the ph at

290 90 The Buffering Effect Acetic Acid Buffer pka = 4.6 CH 3 C CH 3 C - Phosphoric Acid Buffer pka = H 3 P 4 H P 4 - HP 4 - P 4 3- CH 3 C CH 3 C - + H + H P 4- Nearly 00% The maximum buffer capacity In case when the ph = pka The minimum buffer capacity Efficient buffering effect can be obtained when the ph is set within ± the pka.

291 9 Tubing Connections The Effect of Dead Volume on Peak Shapes mau Column Injector Analytical Condition: Column : Inertsil DS-3 (5 μm, 50 x 3.0 mm I.D.) Eluent : CH 3 CN/ H = 5/95, v/v Flow Rate : 0.4 ml/min Col.Temp : 40 Detection : UV5 nm Sample :. Methamidophos. Acephate mau Column Injector

292 9 Cautions on when Cutting the Tubing Vertically cut the tubing therwise, poor peak shapes, poor theoretical plates and cross-contamination may be observed/generated. Do not squash the internal diameter therwise, increase in system back pressure may be observed. Keep the cutting surface as clean as possible therwise, early deterioration of the column may be observed. (Clogging of column inlet filter by impurities or contaminants) Enlarged View Residue samples

293 93 Cutting the Tubing Comparison of Cutting Surface between various Tubing Cutters I.D. 0.5mm Peek Tubing Picture Image by Electron Microscope I.D. 0.5mm Peek Tubing I.D. 0.3mm Peek Tubing Clean Cutter Cat# ) Commercially Available Polymer Tubing Cutter Commercially Available SUS Tubing Cutter

294 94 Effect of Cell Volume on HPLC Analysis The analysis was done using a semi-micro size column mau Semi-micro UV Cell (3. μl) 3. Acetophenone. Benzene 3. Toluene 4. Naphtalene 4 Analytical Condition: Column : Inertsil DS-SP (3 μm, 50 x. mm I.D.) Eluent : A) CH 3 CN B) H A/B = 65/35, v/v Flow Rate : 0.7 ml/min Col.Temp : 40 Detection : 54 nm Injection Vol : μl mau Standard UV Cell (3 μl)

295 95 High Throughput with Column Switching Method If those impurities are eluting later at the end Target Compound Ideally prefer obtaining a chromatogram as follows Impurities Target Compound

296 96 High Throughput with Column Switching Method If those impurities are eluting later at the end Pump Inj. Column Detector Detector Pump Column Inj. Column Pump Waste Switching the valve right after confirming the target compound is introduced to Column. Target Compound Impurities Detector Pump Column Inj. Column Pump Waste General Analysis Method Column Switching Method

297 GL Sciences Inc. HPLC 4. Tips on Maximizing the Performance of an HPLC 97

298 Relative Retention Value 98 Improving Separation using the Mobile Phase The retention time of compounds having ionic functional groups can dramatically change depending on their dissociated and undissociated conditions. The separation or elution pattern can be changed by adjusting the ph value of the mobile phase when the sample contains different/various ionic functional groups. C Analytical Conditions Column : Inertsil DS- 5 μm, 4.6 x 50 mm I.D. H 3 C CH 3 Eluent : CH 3 CN / 50mM Phosphate Buffer = 30/70 Flow Rate :.0 ml/min CH 3 Detector : UV 54 nm Col.Temp. : 40 Sample :. 3,5- Dimethylbenzoic acid (Acidic). Acetophenone (Neutral) 3 NH 3. Benzoic acid (Acidic) 4. Aniline (Basic) 4 C

299 99 3 Improving Separation using the Mobile Phase Analytical Conditions Column : Inertsil DS-3 5 μm mm I.D. Eluent : rganic solvent / 0.% H 3 P 4 Flow Rate :.0 ml/min Detector : UV 84 nm Col.Temp. : 40 4,5 Sulfa drugs ) Sulfamerazine ) Furazolidone 3) xolinic acid 4) Sulfadimethoxine 5) Sulfaquinoxaline 6) Nalidixic acid % CH 3 CN 30% CH 3 CN 40% CH 3

300 Pressure Ratio 300 Comparison between Acetonitrile and Methanol Although Methanol produces high back pressure, it has several benefits Methanol Acetonitrile Solvent Concentration (%) rganic Solvent Cost (HPLC Grade) PRTR Method Toxicity Back Pressure Acetonitrile 3 times expensive than Methanol Applicable High Low Methanol NA Low High

301 30 To Improve Separation Further if the Back Pressure Allows Increase the column length Longer column length offers higher separation efficiency, but with longer analysis time and higher back pressure Usage of smaller particle size Although the back pressure increases significantly, it offers higher separation efficiency maintaining nearly the same retention time 00 mm 5μm 50 mm 3μm 30

302 30 Scaling Down the Column I.D. What are the benefits that can be expected employing such method? Usage of smaller I.D. columns When using smaller I.D. columns, it is a must to adjust the flow rate Resulting in less consumption of mobile phase! I.D. 4.6mm ml/min 60% Less Consumption of Mobile Phase Column Inlet Column utlet I.D. 3.0mm 0.4mL/min

303 303 Expected Results of Scaling Down the Column I.D. Same retention time or separation pattern can be expected by adjusting the flow rate nearly equal to the linear velocity of the mobile phase Analytical Conditions System : L-7400 system Column : Inertsil DS-3 (5 μm, 50 mm) Eluent : A) CH 3 CN B) H A/B = 80/0, v/v. n-butylbenzene Col. Temp. : 40. o-terphenyl Detection : UV 54 nm Injection Vol. : 0μL 3. n-amylbenzene 4. Triphenylene Increase in Sensitivity depending on the detector (UV, PDA, RI, FL etc) mau mau I.D. 4.6 mm (Cross-section area 6.6 mm ) Flow Rate.0 ml/min I.D. 3.0 mm (Cross-section area 7. mm ) Flow Rate 0.4 ml/min

304 Scale Down Limitations of Standard HPLC Systems This should be the limit of scale down when using standard HPLC systems Separation not achieved 5mV I.D. 4.6 mm Flow Rate.0 ml/min I.D. 3.0 mm Flow Rate 0.4 ml/min I.D.. mm Flow Rate 0. ml/min Analytical Conditions Columns : Inertsil DS-3 5μm 50mm Eluent : A)CH 3 CN B) H A/B = 80 / 0, v/v Col. Temp. : 40 Detection : UV 54nm ) Uracil 4) n-butyl benzene ) Caffeine 5) o-terphenyl 3) Phenol 6) n-amylbenzene 7) Triphenylene 304

305 305 System Dead Volume Ideal Actual Actual Theoretical Plates (Separation Efficiency) This difference is very important The difference in plates are caused by the system dead volume. The influence of dead volume gives a major impact to the plates when using smaller I.D. columns or for those compounds that eluted very fast/quickly. Retention Time

306 306 Summary of Maximizing the Performance of an HPLC Set the ph of the mobile phase by referring to the pka of the target compound. Separation pattern can be changed by using different types of organic solvents. The Consumption amount of mobile phase can be reduced by using 3.0 mm I.D. columns or * recycling of the mobile phase. System Dead Volume is another important factor in HPLC analysis, which greatly and directly influences the theoretical plates, sensitivity. * If you re interested in recycling your mobile phase, please visit the following website.

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