HPLC Method Development with Eclipse Plus: Standard Practices and New Columns Agilent Technologies
What are Some Standard Method Development Practices?. Follow preferred method development scheme and do hands-on method development based on selectivity changing parameters i.e. ph, column, or mobile phase types. Use method development software run a few predictive runs and get prediction for best method 3. Evaluate multiple columns/multiple mobile phases in a manual or automated fashion, determine best results (i.e. using valve switching to change between columns, software to track peaks etc.) Practice # is used by many and incorporates hands on learning. It requires nothing special to make it work, so we will work through a method development scheme for this practice.
What Makes a Good Starting Point for Hands-on Method Development?. Choose a C8 column many choices, well known technologies a) A short column minimizes method development time b) Smaller particle sizes provide the resolution needed in a short time c) Newer C8 column choices can improve results based on efficiency, improved peak shape. Choose a simple mobile phase reliable, works with many samples a) Phosphate buffer ph 3, TFA, or formic acid in aqueous portion b) Acetonitrile or methanol as organic modifier 3. Adjust mobile phase to get the desired retention and resolution a) Adequate resolution of all peaks, Rs. requires highest efficiency b) Retention of first peak preferred to be at least k= c) Analysis time below minutes shorter d) Newer column choices, short columns with small particle sizes can provide more efficiency and resolution in a very short time, speeding up method development
Typical Method Development Parameters - Effects of Selectivity, Efficiency, and Retention on Resolution 7. 6. Selectivity Impacts Resolution Most Change bonded phase Typical Method Development Parameters Change mobile phase Plates are easiest to increase 5. Resolution 4. 3. Increase N Increase Alpha Increase k'... R s = N ½ /4 ( -)/ k /(k +) 3 4 5 6 7 8 9 3 4 5 6 7 8 9 Plates: 5 5 5 Alpha:..35.6.85. k :. 4.5 7. 9.5.
Small Differences in α Selectivity - Can Provide Large Differences in Resolution Eclipse Plus C8 Greatest Resolution for this sample α Resolution peaks,.68 6. peaks,3.4 4.97 caffeine sulfamethoxazole barbital 3 4 Eclipse Plus C8 min α Resolution peaks,.33 3.55 peaks,3.3 3.8 Cols: 4.6 x 5, 5 um m.p.: 4% MeOH, 6% water 3 4 Eclipse XDB-C8 min α Resolution peaks,.43 4.5 peaks,3.9 3.55 3 4 min
What Do We Want to Base Our Complete Method Development Scheme On? (Beyond the Start) Changing selectivity to improve resolution with the two key parameters mobile phase and bonded phase Mobile phase ( st choice to change because it s easy) Mobile phase organic modifier (ACN, MeOH etc.) Mobile phase ph over a wide ph range ph - if needed Bonded phase (provides lots of optimization potential) Many choices other than C8 C8, Phenyl, CN AQ for aqueous mobile phases Choose new columns (Eclipse Plus) with best peak shape performance
New Column Choices To Use for Standard Method Development. Shorter columns with smaller particle sizes Rapid Resolution High Throughput Columns (RRHT) with a.8um particle size 5mm for fastest method development Longer columns for maximizing efficiency Many bonded phase choices for a complete method development scheme. Improved bonded phases for superior performance Better peak shape and efficiency result in more resolution Critical to the best methods New Eclipse Plus columns can deliver improved performance
. Start Method Development with RRHT Columns: Different ZORBAX RRHT C8 Bonded Phases for Max Selectivity st choice Best Resolution & Peak Shape nd choice Good alternate selectivity due to non-endcapped 3 4 5 3 3 4 4 Eclipse Plus C8 StableBond SB-C8 Mobile phase: (69:3) ACN: water Flow.5 ml/min. Temp: 3 C Detector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 5 mm.8 um 3 rd choice Good efficiency & peak shape Resolution could be achieved 4 th choice Resolution not likely, Other choices better, for this separation. 3 4 5 3 4 5 3,3 4 4 Eclipse XDB-C8 Extend-C8 3 4 5 min Sample:. anandamide (AEA). Palmitoylethanolamide (PEA) 3. -arachinoylglycerol (-AG) 4. Oleoylethanolamide (OEA) Multiple bonded phases for most effective method development. Match to one you are currently using.
. Agilent Rapid Resolution HT Eclipse Plus C8 Provides Superior Peak Shape and Efficiency mau 8 6 4 ZORBAX RRHT Eclipse Plus C8. x 3 mm,.8 µm 3 bar N: 45 TF:.4 Rs 4,3 :.6 36 % MeCN 64% Water w/.7 ml/l phosphoric acid µl injection nm Flow :.5 ml/min RRHT,.45 ml/min. (Luna) - Hydroxy--(4-Isobutylphenyl) proprionic acid -(3-Isobutylphenyl) proprionic acid 3 Ibuprofen 4 -(4-n-butylphenyl) proprionic acid 5 Isobutyl acetophenone All at.5 mg/ml mau 8 6 4 3 4 5 6 7 8 Phenomenex Luna HST C8 (). x 3 mm,.5 µm 6 bar N: 38 TF:. Rs 4,3 :.47 min 3 4 5 6 7 8 ZORBAX RRHT has at least 8% more efficiency than Luna HST C8() That means greater than baseline resolution when needed. min
. Eliminate Tailing and Maximize Resolution with Eclipse Plus Columns Mobile Phase: 65% ACN: 35% 5 mm phosphate buffer (ph 7.4) Tf =. Eclipse Plus C8, 4.6 x 5mm. Nortriptyline. Dipropylphthalate Competitive C8, 4.6 x 5mm Tf = 4.86.5.5 Superior peak shape and better selectivity with Eclipse Plus means more resolution, easier quantitation and better results in your separations. min
. Eliminate Tailing and Maximize Resolution with Eclipse Plus Columns Trace level Analytes Competitive C8 4.6 x 5 N =,5 Tf =.43 Eclipse Plus C8 4.6 x 5 N =, Tf =.95 Note that both columns have the same efficiency and are well packed but the improvement in tailing factor, and the selectivity advantage that creates, provides better resolution of trace level compounds
. Choose New Eclipse Plus C8 for Superior Efficiency and Peak Shape Analysis on Other C8 Poor sensitivity Due to broad peaks Peaks do not tail, but are distorted. Analysis on Eclipse Plus Better Sensitivity Sharp, Efficient Peaks Excellent peak shape
How Do We Implement These New Column Choices in the Method Development Process? Follow a method development process around standard practices ONLY when the first steps allow you to incorporate new products for fast, good results! A method development scheme based on simple strategies for optimizing selectivity and resolution can incorporate new products. The 3 key parameters must be: mobile phase mobile phase ph bonded phase
First Steps in a Method Development Scheme Choose Eclipse Plus C8 Low ph Adjust %ACN for.5 <k < Band spacing problems Change % organic Band spacing problems Change organic modifier Adjust % organic for.5 < k < Band spacing problems STEP STEP STEP 3 STEP 4 Change bonded phase Eclipse Plus C8, SB-C8, CN, Phenyl, other RP Select a high quality (Eclipse Plus) C8 or C8 bonded phase first for good retention and resolution with typical acidic, basic and neutral samples. Optimize the organic component of the mobile phase to change selectivity Choose alternate bonded phases to completely optimize method if needed Choose SB-C8 for ph - See New Poster Pub No. 5989-78EN With new RRHT column choices, these steps can be done quickly, making it possible to truly find the best method.
See Pub No. 5989-78EN for Complete Poster
Let s Look at Method Development of These Compounds propranolol pindolol dipyridamole β-blocker Anti-arrhythmic Vasodilator Ca+ channel blocker disopyramide diltiazem
Start at Low ph, Adjust Organic Eclipse Plus C8 Cardiac Drugs with Acetonitrile mau 4 3 mau 5 75 5 5 mau 6 4 - Column: ZORBAX RRHT Eclipse Plus C8, 4.6 x 5 mm,.8 m Mobile Phase: A: 5 mm NaH PO 4, ph 3. B: ACN Flow Rate:. ml/min Temperature: 3 C Detection: UV 4 nm Sample: Cardiac Drugs.Pindolol. Diisopyridamide 3.Propranolol 4.Dipyridamole 5. Diltiazem 4% ACN 3% ACN RRHT Eclipse Plus C8 allows for very fast optimization of % organic in mobile phase. % ACN Good resolution Fast analysis No time wasted To get this k on a 5cm column at ml/min would require.5 hours run time!! k= 44!! 4 6 8 min
Mobile Phase Optimization on Short RRHT 4.6 x 5 mm, SB-CN Column mau 5 5 mau 5 5 5 5 3 ) 3 4 5 3 4 5 3 4 5 6 6%B Rs 4,5=.96 6. Estril. Estradiol 3. Ethynyl Estradiol 4. Dienestrol 5. Diethylstilbestrol 6. Ethynl estradiol methyl ester 55%B A: Water B: 3 % MeOH/7 %MeCN µl injection, nm Rs 4,5=.59 mau 5 5 3 4 5 5%B Rs 4,5=3.9 6 5 3 4 5 6 7 8 RRHT columns allow for very fast optimization of % organic in mobile phase. min
Comparison of Optimal Conditions On Columns with Different Dimensions Length, Particle Size, Resolution mau 8 6 4-5 5 ) mau 4 4 3 5 3 4 RRHT SB-CN 4.6 x 5 mm,.8um.5 ml/min µl injection 6.5 5 A: Water 5 RRHT SB-CN 4.6 x mm,.8um.5 ml/min µl injection 6. Estril. Estradiol 3. Ethynyl Estradiol 4. Dienestrol 5. Diethylstilbestrol 6. Ethynl estradiol methyl ester B: 3 % MeOH/ 7 %MeCN 4 % B Detection: UV nm mau 75 5 5 75 5 5 ).5 5 7.5 4 3 5 SB-CN 4.6 x5 mm, 5um. ml/min 3 µl injection.5 5 7.5.5 5 7.5 RRHT Columns allow rapid resolution optimization during initial method development. Column lengths compared quickly. 6 min
Why Do We Recommend Changing Organic Modifier Before Changing the Bonded Phase? It s easy acetonitrile and methanol are readily available! It works on any bonded phase, so it gives you a tool to optimize a separation no matter the column choice!
Start at Low ph on Eclipse Plus C8 RRHT Cardiac Drugs with Methanol, Changing Organic Modifier mau 4 3 mau 5 75 5 5 Column: ZORBAX RRHT Eclipse Plus C8, 4.6 x 5 mm,.8 m Mobile Phase: A: 5 mm NaH PO 4, ph 3. B: MeOH Flow Rate:. ml/min Temperature: 3 C Detection: UV 4 nm Sample: Cardiac Drugs.Pindolol. Diisopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole 7% MeOH 5% MeOH Adjusting MeOH influences retention like adjusting ACN Solvents can be compared quickly with RRHT columns MeOH changes selectivity and gives longer analysis time for these analytes mau 8 6 4 4% MeOH 4 6 8 k= 49!! min
Method Development - Change Organic Modifier Comparison of Acetonitrile and MeOH mau 4 8 6 4 - mau 4 8 6 4 - Column: ZORBAX RRHT Eclipse Plus C8, 4.6 x 5 mm,.8 m Mobile Phase: A: 5 mm NaH PO 4, ph 3. B: organic Flow Rate:. ml/min Temperature: 3 C Detection: UV 4 nm Sample: Cardiac Drugs.Pindolol. Disopyridamide 3.Propranolol 4.Diltiazem 5. Dipyridamole 5 3 3 4 3 4 5 3% ACN 5% MeOH Resolution of pairs (,3), (3,4), (4,5) is better in MeOH. Peak 5 selectivity shift Resolution of critical pair (,), is better in ACN
Change Mobile Phase - Acetonitrile vs. Methanol on ZORBAX Eclipse XDB-CN with Estrogens Column: ZORBAX Eclipse XDB-CN Column Dimensions: 4.6 x 5 mm, 5 m Mobile Phase: As shown Flow Rate:. ml/min Injection Volume:. l Column Temperature: 5 C Detector: UV, nm 3 4 5 Mobile Phase: 35:65 ACN:Water Solvent comparison with all samples can yield optimized methods. 6 7 Sample:. Estriol (.3 g/ l),. -Estradiol (.3 g/ l), 3. Ethinyl Estradiol (.47 g/ l), 4. Dienestrol (.3 g/ l), 5. Diethylstilbestrol (.8 g/ l) 6. Ethynylestradiol 3-methyl ether (.3 g/ l) 7. Ethynodiol Diacetate (.39 g/ l) 4 6 8 4 6 8 min 4 Mobile Phase: 47.5:5.5 MeOH:Water 3 5 6 7 4 6 8 4 6 8 min
Why Is Changing the Bonded Phase Effective? Differences in interactions between polar and non-polar compounds. Other types of interactions with a bonded phase can be exploited (pi-pi interactions etc.) These all change with bonded phase! Changing the bonded phase can improve selectivity/resolution, reduce analysis time When you use RRHT (.8um) columns the comparison of bonded phases can be done quickly!! You must have multiple RRHT column choices available
Bonded Phase Selectivity Differences in 3% ACN mau mau mau mau RRHT SB-CN 4.6 x 5 mm,.8 m RRHT SB-Phenyl 4.6 x 5 mm,.8 m RRHT SB-AQ 4.6 x 5 mm,.8 m RRHT Eclipse Plus C8 4.6 x 5 mm,.8 m 5 Different bonded phases compared Analysis time of each run is only minutes Comparison done in optimum % organic The fast runs mean a comparison can be done even if you have a good separation on the C8 More chances to optimize! mau RRHT SB-C8 4.6 x 5 mm,.8 m
Selectivity/Bonded Phase Choices: Eclipse Plus C8 vs. C8 and RRHT vs. RR RRHT Eclipse Plus C8, 4.6 x 5mm,.8 um. Oxybenzone. Internal Std. 3. Octylmethoxycinnamate 47 bar N: 88 Pw:.4 min. TF:. N: 6 Pw:.47 min. TF:.6 N: 8 Pw:.93 min. TF:. ) 3 4 Rapid Resolution Eclipse Plus C8, 4.6 x 5mm, 3.5 um N: 5 Pw:.9 min. TF:.9 N: 66 Pw:.37 min. TF:.99 N: 63 Pw:.6 min. TF:.98. min. 3 4 min 88 bar Conditions: Mobile Phase.: Water: Acetonitrile (3:7) Flow Rate:. ml/min. Detection: UV 3nm Temperature: 3 C Sample: Lip balm extract in ACN (melted at C ACN, cooled and.45 um filtered) Less retention can save significant time the C8 is a good choice here. The RRHT column is delivering the efficiency and resolution expected, but the C8 bonded phase may be the best choice. min
Selectivity of Polar Phases Provides Optimum Separation of Steroids Versus Non-Polar C8/C8 3 4 5 6 7 5 5 4,5 Column: ZORBAX Eclipse XDB-Phenyl Mobile Phase: 48:5 ACN:Water 3 6 7 5 5 5 Column: ZORBAX Eclipse XDB-C8 3 4 6 Column: ZORBAX Eclipse XDB-CN Mobile Phase: 35:65 ACN:Water Mobile Phase: 54:46 ACN:Water 5 5 4 3 5 Column: ZORBAX Eclipse XDB-C8 Mobile Phase: 6:4 ACN:Water 6 7 5 5 7 min Column Dimensions: 4.6 x 5 mm, 5 m Flow Rate:. ml/min Injection Volume:. l Column Temperature: 5 C Detector: UV, nm Sample:. Estriol (.3 g/ l),. -Estradiol (.3 g/ l), 3. Ethinyl Estradiol (.47 g/ l), 4. Dienestrol (.3 g/ l), 5. Diethylstilbestrol (.8 g/ l) 6. Ethynylestradiol 3-methyl ether (.3 g/ l) 7. Ethynodiol Diacetate (.39 g/ l) Eclipse XDB-CN is the optimum bonded phase the only one that can resolve all the components in under minutes.
So then Why is ph Critical to Method Development? What compounds are ph sensitive? How does ph affect retention and resolution? How does ph influence method development? How does ph influence column choice?
When Does ph affect Resolution? Compound Type Comparison non-polar polar ionizable naphthalene benzyl alcohol benzoic acid ph 3 acenaphthene nitrobenzene benzanilide 3 4 5 6 7 8 9 3 4 5 6 7 8 3 4 5 6 7 8 ph 7 3 4 5 6 7 8 9 3 4 5 6 7 8 3 4 5 6 7 8 Time (min) Time (min) Time (min) Ionizable compounds acids and bases can change retention and selectivity most with changes in ph
Change in Retention with ph for Ionizable Compounds is Key to Method Development Non-charged analytes have better retention (i.e. acids at low ph and bases at high ph) Silanols on silica ionize at mid-ph, increasing retention of basic analytes (i.e possible ion-exchange interactions) Choose mobile phase ph to optimize retention and selectivity during method development Eclipse Plus can be used over a wide ph range Other choices exist for high ph
Change in Retention with ph for Ionizable Compounds is Compound Dependent Non-charged analytes have better retention (i.e. acids at low ph and bases at high ph) retention time (min) 8 6 4 Acetylsalicylic acid Pyridine Codeine Procainamide Amphetamine Caffeine pka 3.5 pka 5. pka 8. pka 9. pka 9.9 pka 4 Mobile Phase: 45% MeOH: 55% mm Phosphate Buffer ph.5 ph 6.5 ph 8 ph.5 ph
Retention vs. ph for Basic Antihistamines - Less Variation in Retention at Low ph 5 4 Column: Eclipse XDB-C8 4.6 x 5 mm, 5 m Mobile Phase: 75% 5 mm phosphate buffer 5% Acetonitrile Doxylamine Chlorpheniramine Triprolidine Diphenhydramine pka 4.4, 9. pka 9. pka 6.5 pka 9. Retention Volume ml 3 No change in retention in this range 3 4 5 6 7 ph Slight changes in ph may dramatically change retention and selectivity Always evaluate retention changes with ph during method development
Therefore ph Should be Considered a Key Method Development Parameter A single column can be used over a wide ph range making selectivity comparisons quick and reliable when ph is changed. May have better selectivity in the ph range 3 8. Other bonded phases can be reconsidered for mid ph, just like at low ph
Method Development Scheme Adding Mid ph From low ph ZORBAX Eclipse Plus C8 ph 7 (6-9) - 5 mm buffer, Adjust %ACN for.5 <k < STEP 5 Band spacing problems STEP 6 Change % organic Band spacing problems STEP 7 Change organic modifier (MeOH) Adjust % organic for.5 < k < Restart at STEP 6 Band spacing problems STEP 8 Try Eclipse Plus Phenyl-Hexyl, Eclipse XDB-CN, Phenyl or Bonus-RP Restart at STEP 5 Mid ph can provide better selectivity It may be more compatible with your sample The process for investigating mid ph is the same as for low ph Eclipse Plus delivers outstanding performance at mid ph Alternate bonded phases should also be considered if improved selectivity is desired
Eclipse Plus Columns Are Ideal for Method Development at Low and Mid ph Can be used from ph -9 Accommodates method development in low and mid ph ranges Allows ph sensitive separations to be optimized Excellent peak shape and efficiency achieved with new Eclipse Plus material
Eclipse Plus Can Be Used Over a Wide ph Range (ph 9) to Optimize Selectivity ph.7.% formic acid/acn 3 4 5 6 7. procainamide. buspirone 3. pioglitazone 4. eletriptan 5. dipyridamole 6. diltiazem, 7. furosemide 4 6 8 ph 4.8 NH 4 AC/acn 6 min Selectivity and resolution can change with ph 7 5 3 4 4 6 8 ph 7 NaPO 7 4 /acn 3 6 min Eclipse Plus can be used with many mobile phases and ph s 4 5 4 6 8 Conditions: Column: Eclipse Plus C8 4.6 x mm, 5um Gradient: 9% in minutes Detection: UV 54 nm
Selectivity Differences at ph and ph 7 Can Be Dramatic Eclipse Plus C8 4.6 x 5mm, 5 um. Acetaminophen. Caffeine 3. Acetylsalicylic acid 4. unknown 3 ph.7 4.5.5.5 3 3.5 3 4 min. ph 7. min Column: 4.6 x 5, 5 um PN 959946-96 Gradient: -6% B/3 min. ph.7: A:.% formic acid B:.% fa in ACN ph 7.: A mm Na phosphate B: ACN Sample: generic Excedrin tablet.5.5.5 3 3.5 min Selectivity can change dramatically from ph 7 with many samples, such as this analgesic tablet. Eclipse Plus can be used under both conditions
Selectivity Differences at Mid ph Key to Resolution Eclipse Plus Performs well at Low and Mid ph Eclipse Plus C8 4.6 x 75 mm 3.5 µm mm phosphate buffer, ph 3. 3 N=57 TF:.8 3 4 5 6 7. Famotidine. Cimetidine 3. Pirenzipine Eclipse Plus C8 4.6 x 75 mm 3.5 µm mm phosphate buffer, ph 7. N=8 TF:.99 3 Columns: as listed Mobile Phase: % MeOH, 8% mm phosphate, ph as listed Flow Rate: ml/min. Detection: UV 3 semi micro flow cell N=79 TF:.5 min 4 6 8 4 6 For this sample resolution is not possible at ph 3, but is at ph 7 Eclipse Plus was used to experiment at both ph s min
Try an Alternate Selectivity at Mid-pH if Needed One choice for alternate selectivity is Bonus-RP. The alkyl-amide bonded phase provides very different selectivity at mid-ph Bonus-RP Eclipse XDB-C8 3 3 4 4 Mobile Phase: 75% mm NaCitrate, ph 6 5% MeOH Flow Rate: Temperature: Detection: Injection Vol: Sample:. ml/min Ambient UV 54 nm 3 L Cephalosporins. Cephalexin. Cephaclor 3. Cephuroxime 4. Cephoxitin 4 6 8 Time (min) Mid ph was the second ph region to consider. High ph can also be considered if low or mid ph did not resolve all analytes.
Method Development at High ph a Third Choice From Mid ph ZORBAX Extend-C8 STEP 9 ph.5 (9-) 5 mm ammonia, or TEA, or 5 mm organic or borate buffers T = 5 C (ambient 4 C) Adjust MeOH for.5 <k < Reasons to Consider High ph Increase retention of basic compounds by analyzing them in noncharged form Improve selectivity Band spacing problems STEP Change organic modifier (ACN or THF) Adjust for.5 < k< Try different HPLC mode
High ph Increases Retention of Antihistamines Extend-C8 Column: ZORBAX Extend-C8, 4.6 x 5 mm, 5 m Mobile Phase: See Below Flow Rate:. ml/min Temperature: RT Detection: UV 54 nm Sample:l:. Maleate. Scopolamine 3. Pseudoephedrine 4. Doxylamine 5. Chlorpheniramine 6. Triprolidine 7. Diphenhydramine ph 7 3% mm Na HPO 4 7% MeOH,3 4 5 7 ph 3% mm TEA 7% MeOH 3 t 5 R = 8.5 t R =.4 4 7 6 6 5 Time(min) 5 Time(min) The retention of this sample of basic compounds increases at high ph.
Conclusions New column choices, like.8um RRHT columns and Eclipse Plus bonded phases can enhance typical approaches to method development. These columns provide fast analysis, allowing more options to be considered quickly, and with superior performance the best results are readily achievable. Since many compounds are ionizable, and retention and selectivity of these compounds are greatly impacted by ph, a method development scheme for LC methods should encourage varying ph. Any method development scheme should also include the use of different bonded phases. Different bonded phases and columns can be selected by ph as well. The third key aspect of method development is mobile phase and organic modifier and this is an effective tool for many separations.