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HPLC conditions for basic compound?Chromatography Forum: LC Message Board: HPLC conditions for basic compound?
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By Anonymous on Tuesday, June 1, 2004 - 06:16 pm:
Phil,
Make a Google search on "Tswett" and "Chromatography" keywords or read about history of chromatography on
http://www.chromatographyonline.com/lcg ... rticle.pdf
It was invented by a Russian scientist Tswett in 1903. His name "Tswett" on Russian means "Color". People think that he's put his name in the method's name "Chroma"tography. Actually, first chromatographies looked like a set of colored bands (leaf pigments)
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By Anonymous on Saturday, June 5, 2004 - 09:30 am:
Or that he used the colored bands as an excuse to work his name in!
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By Kazimierz on Thursday, July 1, 2004 - 03:18 pm:
Separation of basic compounds:
1. On silica column with nonequous methanol + 0.02-0.1% of HClO4 Flanagan R.J. et al. J. Chromatogr. 247, 15-37, 1982 (amiodarone, DEA)
2. On silica column with methanol:H20 (antipyrine)
3. On Synergy-Polar-RP or Luna C18 (AcCN:H20)2.8%
+ 0.2% HFBA
4. On silica Itertsil: Hydrophilic Interaction LC
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By Kaoula on Friday, July 2, 2004 - 07:08 am:
Hi ALL:
Please i need a clarification about the rule for choice between L7 coulmn and L1 coulmn ??
thank you ver match
Kaoula
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By jack on Wednesday, July 14, 2004 - 07:54 pm:
is anyone have some idea to extract streptomycin in honey matrix and then run on lc/ms/ms, please help
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By Anonymous on Thursday, December 19, 2002 - 09:37 am:
i have a basic compound to run on HPLC, basically the structure is 2 fused rings with primary, secondary, and 3rd-order amine attached or fused into the rings, and a hydroxy group also attached to the ring, the 1st scout run with gradient water/ACN/TFA on C18 column didnot get any good result of course, and the peak didnot retain on column at all. the 2nd try i put an c18-aqua with high percent(98%) water at start, no good; 3rd try i use sodium bicarbonate at pH 8 on the C18 or C18-aqua column, still not retain at all if water decreased to 96% at start.
then i use water/ACN/0.1% TEA with non silica column which is zirchrom hydrocarb column, since the pH with 0.1%TEA is about 11. i still get messy baselines and big broad peak, the sample run can not distinguish from the blank.
anyone have good ideas for a good set of conditions for this?
since the blank run on TEA is also messy, maybe i should try DEA instead?
appreciate any advice.
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By Einar Pontén - SeQuant AB on Thursday, December 19, 2002 - 11:14 am:
I suggest that you try HILIC mode of separation.
The ZICâ„¢-HILIC column is well suited for basic compounds.
http://www.sequant.com
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By Chris Pohl on Thursday, December 19, 2002 - 12:13 pm:
I would suggest trying cation exchange. Since you are reporting insufficient retention via reversed phase even in the presence of high water content eluents, the sensible strategy would be to accomplish retention based on the analyte's fundamental property: its cationic nature. Most likely separation can be accomplished using any of the many available silica or polymer based materials, although the tendency of your analyte to strongly interact with silanols may compromise chromatographic performance on silica based materials. Alternatively, one can use any of a number of available polymer materials. One material specifically designed for such analytes is the PCX-100 column (http://www.dionex.com/app/tree.taf?asset_id=9324). Best performance is generally achieved for analytes of this sort with potassium or better still cesium based eluent systems containing 20% acetonitrile.
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By Daren on Thursday, December 19, 2002 - 01:09 pm:
I also think HILIC is good idea, but if you are looking to stay with reverse phase you might want to try a more polar stationary phase. A good choice for your application might be the Waters Xterra Phenyl column. You can run it in 100% aqueous if needed. I would stick with TFA and keep the pH low (around 2),you may get added retention from ion-pairing interaction of TFA.
Since you have so many amine functional groups with varying pKa your going to have trouble getting a sharp peak once you start raising the pH.
Daren
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By Daren on Thursday, December 19, 2002 - 01:13 pm:
you can always throw in an ion-pairing agent at low pH like octane sulfonic acid and go back to the C18. But I tend to find ion-pairing methods not very robust.
Daren
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By Einar Pontén - SeQuant AB on Thursday, December 19, 2002 - 01:28 pm:
Beside the separation problem I think you should also consider what different detection techniques you may need today and later on.
If MS, then ion-pairing and traditional ion-exchange may be less suitable.
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By Uwe Neue on Thursday, December 19, 2002 - 03:52 pm:
There are several possibilities to solve the problem. First, you can use one of the columns that have been optimized to give good retention in 100% (!!!) water (do not use 98% or 96% as you did). A column that might be useful for you is the Atlantis d-C18 column from Waters that has been designed for this kind of difficulties.
You can also go to a higher pH, but I would not use a carbon column. You can use one of the newer C18 zirconia columns, or an XTerra MS C18 with ammonia or ammoniumbicarbonate at pH 10 as a mobile phase. You can also change the retention pattern completely, and use hydrophilic interaction chromatography combined with ion exchange. In order to do this, you should use a silica column. They are nicely retentive, and they do not give tailing peaks, since there is no bonded phase to hinder the interaction with the silanols. Considering the description of your compound, I believe that the last approach will work best: you will have plenty of retention, you just need to get used to how to manipulate retention in this mode of chromatography. I can get you some advice, if needed.
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By Michael Goodwin on Friday, January 10, 2003 - 03:04 pm:
I have a similar situation and thus posting it here instead of as a new question.
I am developing a method for quantitation of a small peptide (~650Da) that has 4 amine groups. The compound is provided in a disulfate salt form.
On reverse phase columns (beta basic C8 Hypersil, C18 phenomenex luna2, Discovery F5) the compound is unretained in all mixtures of water/acetonitrile each w/ 0.1% v/v formic acid.
The compound is added to 0.1M KOH to attempt desulfating and return to free base form. Still no retention.
On Silica, only high levels of water:MEOH elute compound, and does so quickly. When compound is added to mouse serum matrix, the matrix co-elutes and interferes with detection (DAD at 269nm, also have ES-MSD available)
I have switched to a PRP-X200 SCX 2.1mm x 15cm 10u particle column that has low capacity exchange sites.
mobile phase is:
A = Water w/ 0.1% v/v TFA
B = solvent A w/ 500mM NaCl
C = Acetonitrile
flow = 0.5 ml/min
With the TFA and NaCl, I have stopped adding the peptide sulfate into KOH and just inject it as is.
I am able to retain the compound and elute it, however I am experiencing a lot of peak broadening.
Some examples include:
mobile phase A:B:C = 45:30:25 k'=18.9 w/ 50% peak ht width (pkw)= 3.1 minutes
A:B:C = 30:45:25 k'=3.8 pkw = 1.8 min
A:B:C = 0:75:25 k'=0.67 pkw = 0.2min
A:B:C = 40:30:30 k'=3.32 pkw = 2 min
A:B:C = 20:30:50 k'=1.5 pkw = 1.3min
Essentially, if I increase acetonitrile concentration or salt concentration the peak gets narrower, but retention suffers greatly which will increases the chance that matrix components will coelute. Peak shape only looks good when compound is essentially unretained.
Decreasing organic or salt gives better retention with unacceptable peak broadening.
test sample concentration is 10uL injection of ~0.5ppt compound. 0.05ppt concentration does not improve peak broadening so I do not believe I'm saturating the exchange sites. The problem persist with 0.2 and 1 ml/min flow rates.
Any ideas on how to get retention with narrow peak width?
I'm thinking next to try the above suggesstion of purchasing octane sulfonic acid to take advantage of the RP character of the PRP-X200 column.
Or, are the amine groups binding to silanols on the PRP column and perhaps it needs some end capping agent??
Any suggestions or explanations will be greatly appreciated.
Thank you.
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By Uwe Neue on Friday, January 10, 2003 - 04:05 pm:
You have an interesting problem.
The PRP column is not based on silica, and therefore does not have silanols. The broad peaks are most likely due to the fact that you are using a polymeric packing. These packings tend to give broader peaks.
If I understand this correctly, you ultimately want to determine your compound in a mouse serum matrix. Let me propose to you an approach that is similar to the ideas that Naidong Weng has published, and that I am currently excited about. I believe it was published in the Rapid Communications in MS, if I remember correctly.
Let us first worry about the HPLC separation, then let us discuss the sample preparation. I recommend to use a silica column in HILIC/ion-exchange mode. You have had very good success with your standard. Your compound is retained on silica in methanol-water. You can manipulate this retention by changing the methanol concentration (more methanol = more retention) or with pH (silica is a weak cation exchanger). You should end up with a mobile phase with a fairly high methanol content.
For the sample preparation, I would now try to do an acetonitrile precipitation first. Dilute your serum sample 5:1 with MeCN! This precipitates the proteins, and you can inject the supernatant directly into your HILIC HPLC. If the stuff is still too dirty, you need to do SPE for sample clean-up. I would use a cation exchange method. Load your sample onto an Oasis MCX SPE device, wash with water/acid, then with methanol/acid, then elute with a high concentration of triethylamine in methanol. You can find details of the procedure on the Waters web side, but you may need to increase the concentration of base to get your sample off (I expect it to be strongly retained on the ion-exchanger since you said it has 4 amino groups).
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By Uwe Neue on Saturday, January 11, 2003 - 04:03 pm:
The Weng Naidong paper is in Rapid Communications in MS 2002; 16; 1965-1975
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By Michael Goodwin on Monday, January 13, 2003 - 10:43 am:
Thank you Uwe. I'll try out the suggestions and post any progress.
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By Einar Pontén - SeQuant AB on Monday, January 13, 2003 - 03:14 pm:
The small and basic tripeptide - Gly-His-Lys - was retained on a ZICâ„¢-HILIC column (high conc acetonitrile). This tripeptide showed more tailing than the basic polypeptides;
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe and
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg.
When the ion strength was increased the tailing for Gly-His-Lys decreased, while k' only changed from 5.8 to 4.6. The polypeptides (k' 2.2 and 3.4) were less affected.
Thus, in this case the zwitterionic functional group showed a selectivity based on both charge and hydrophilicity. The ion exchange mechanism was suppressed by a higher ion strength in the mobile phase.
I suppose that your "small peptide (~650Da) that has 4 amine groups." would behave as the polypetides mentioned above.
I agree with the sample preparation procedure suggested by Uwe Neue. If too high concentrations of the amine is required, then a ZICâ„¢-HILIC SPE could be an alternative.
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By Chris Pohl on Tuesday, January 14, 2003 - 12:14 pm:
Michael Goodwin,
Regarding your original question, a point to keep in mind is that the nature of your eluting cation is an important consideration in terms of obtaining good peak shape with some polymeric stationary phases. I'm not sure exactly how PRP-X200 is sulfonated but it's likely that this has produced some relatively sluggish mass transport. Potassium based eluent systems typically significantly improve chromatographic performance under these conditions and cesium based eluent systems will work even better.
In addition, contrary to the comments above by Uwe, modern polymeric stationary phases are commonly comparable in chromatographic efficiency to the silica based materials of the equivalent particle size with the added advantage of much better hydrolytic stability. Another option worth considering is a switch to a pelicular cation exchange material such as the PCX100, available from Dionex. This material provides excellent peak shape for a variety of cationic analytes which prove problematic on silica based materials.
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By Anonymous on Wednesday, January 15, 2003 - 10:27 am:
Chris,
what do you mean with cesium based eluent systems?
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By Chris Pohl on Wednesday, January 15, 2003 - 03:44 pm:
Anon above,
Cesium based eluent systems provide superior chromatographic efficiency in polymeric cation exchange systems. In the example above, the dominant eluent cation was sodium (from the sodium chloride). Substituting cesium chloride under the same conditions will produce significantly better performance. Keep in mind, since cesium is a more potent eluent cation, it will be necessary to adjust the concentration of the cesium accordingly.
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By Uwe Neue on Wednesday, January 15, 2003 - 04:15 pm:
Chris,
I have little experience with ion exchange, but we have looked at most modern reversed-phase columns, including polymeric packings. None of the porous polymeric ones had mass transfer coefficients that reached a porous silica-based packing. Of course, you can argue that non-porous packings have a better mass transfer coefficient, but due to the compromises involved in these packings, none of them is anywhere close to where they should be.
Close, but no cigar!
Best regards
Uwe
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By Benjamin on Thursday, January 16, 2003 - 06:04 am:
Chris, Uwe;
Over 20 years I have tried to use polymer-based columns of all types. Everytime I develop a new method or separation I always consider their use, many times I have tried them, and almost invariably, I end up going back to silica ones.
I think it is fair to say that polymer ones just do not have the efficiency we would like to see. I have yet to obtain a decent reversed-phase application using them. However, I have obtained some use from them in ion-exclusion and ion exchange.
Benjamin
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By Chris Pohl on Thursday, January 16, 2003 - 09:26 am:
Uwe, Benjamin,
First of all, I'm afraid this discussion has devolved into personal opinion dominating over relevant factors. The fact is, in my 30 years of experience in HPLC and Ion Chromatography I've had a chance to use a lot of different materials for separation of ions. Whether or not a polymeric material has comparable efficiencies, higher efficiencies or lower efficiencies depends upon the analyte and design of the stationary phase. It is not useful to generalize about the relative merits of polymeric and silica based phases based on generalizations surrounding efficiency. The fact is that selectivity is the dominating factor in stationary phase design and in my experience polymeric phases afford much more selectivity control than silica based materials. It really doesn't do any good if a particular column has more theoretical plates than another if the job at hand requires, say resolution of bromate and chloride, and the two analytes coelute on the column with the highest efficiency. Furthermore, polymeric columns are typically considerably more stable. A silica column that last six months is doing well but I have seen data on polymeric columns that have been in use for five years at pH 13 with no significant degradation. Perhaps, it's all just a matter of personal opinion. The preceeding probably will not convince you. So, can we just agree to disagree?
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By Benjamin on Thursday, January 16, 2003 - 11:25 am:
Chris;
I agree to disagree to agree (just kidding). Basically I feel that your comments agree with mine. Polymeric materials are more adequate for the analysis of ions (ion exclusion, ion exchange). But as Uwe says and I mentioned, the same type of materials do not perform as well in reversed-phase separations. Even if silica-based columns are not very long-lasting, they are still the best option in many cases.
I believe you have polymeric materials that have been used for 13 years or more. Please also believe me that I have some, older than 10 years, and I have not been able to obtain any good results from them.
Benjamin
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By Michael Goodwin on Thursday, January 23, 2003 - 02:07 pm:
Still have some work to do with these suggestions, but I figured I should chime in and let everyone know that I'm still around and listening.
I'll post more details when the study is complete, but here's the short of it.
Switched to HILIC using ammonium formate/NaCl and Methanol and had difficulty eluting the compound off the column.
Briefly switched to PRP-X200 using CsCl instead of NaCl.
Compound eluted quicker, but still alot of peak broadening.
Switched to HILIC again with CsCl and seeing promising results. I have a good feeling about this combo and adjusting CsCl in the mobile phase may give the right optimization. More to come.
I also found the discussion of polymer vs. silica interesting.
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By Einar Pontén - SeQuant AB on Thursday, January 23, 2003 - 02:17 pm:
Yes, the agree-to-disagree dialog is intereting.
Michael,
Why don't you send us a sample and have the problem solved....?
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By Anonymous on Friday, January 24, 2003 - 07:41 am:
What is more soluble in MeCN? CsCl or NaCl?
Thanks
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By Chris Pohl on Monday, January 27, 2003 - 01:03 pm:
CsCl is considerably more soluble in organic solvents than NaCl although in both cases the solubility is pretty low unless some water is present.
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By Michael on Friday, May 23, 2003 - 12:34 pm:
This has been concluded for awhile, but I figure better late than never. The final method for these extremely polar peptides:
Silica column 5u 4.6mm * 250mm in HILIC mode:
mobile phase: 75% (.1%v/v acetic acid with 3mM CsCl) and 25% v/v MeOH isocratic flow 1.0 ml/min
Preparation: 1:1:5:13 serum:internal std (aq w/ acetic acid):water with .1% acetic acid: acetonitrile (added last)
acetonitrile ppt out serum components and leaves enough internal standard and sample for quantitation with uv detector at 269nm. samples are also centrifuged 5 min at 4000RPM to reduce turbidity.
CsCl and acetic acid worked wonders over NaCl.
peptide of interest elutes with baseline resolution as two peaks (can now resolve both components) with k'~ 0.9 & 1.0 peak width ~0.2min. Internal standard (same structure, one less hydroxyl group), also possesses baseline resolution. All three components have great peak symmetry with no obvious tailing.
Serum peaks virtually non-existent and elute in void volume.
I truly appreciate all the suggestions provided.
thank you
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By CHVS NAGARAJU on Wednesday, July 30, 2003 - 05:31 am:
What is more soluble in MeCN? CsCl or NaCl?
Solubility is depend on the polarity of the solute and solvent. Based on 'Like solves like' concept Cesium chloride is more soluble than Sodium chloride in Acetonitrile(CH3CN).
As per the elotropic series(Silica based), n-hexane is having zero polarity where as water maximum.
Acetonitrile's polarity is 5.8
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By Andreas Neumaier on Friday, August 1, 2003 - 07:48 am:
To get back to Anon who started the tread:
You are using TFA with a basic compound so you are getting an very polar ion pair which don't retain (much) on typical C18. The lower the pH of the mobile phase the lower the retention time.
When you use a fluorinated phase (Fluofix, Fluorophase RP or Fluosil or any other column aviable) the TFA-ion pair will be retained quiet good on the fluorinated phase (even pure TFA is retained). And (from my experience) the funny part of it is: basic compounds with lots of basic N "bounds" more TFA and is retained stronger on a fluorinated column.
With other words: Using a fluorinated phase and TFA with a compound (which is able to build TFA-complexes or TFA-ion pairs) is one of the simplest ion pair systems and the mobile phase is very easy to mix (just add an exactly volume of TFA to the mobile phase).
I have run into some problems with fluorinated phases (some of them happens with ALL silica columns):
- Never let TFA inside the column when it isn't used. Flush the column every time it won't be used for some time (hours).
- Tailing with basic compounds can be connected to the concentration of TFA in the mobile phase. Double the amount of TFA and check tailing again.
- Keep an eye on the pH of the mobile phase. It is possible to operate even at low pH (2 to 2.5) for some time when the column is flushed after the last injection.
- Check the UV absorbance of the mobile phase when operation at low wavelengt. TFA is strongly absorbing at low wavelenght (bye bye linearity...).
- When ordering a fluorinated phase, take at least two different batches. If the columns differs too much, send them back, take your money and change to an other manufacturer.
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By Anonymous on Thursday, August 21, 2003 - 11:41 am:
Hi everyone
Please suggest HPLC conditions for the determination of cyclosporin A related substances.
Thanks
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By Anonymous on Friday, November 28, 2003 - 08:46 pm:
Soy estudiante de maestrÃa en ciencias animales, quiero evaluar los componentes de la fracción etanólica de hojas de Ambrosia cumanensis, por medio de HPLC. Qué me recomiendan para tener en cuenta en el procedimiento?
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By Einar Pontén - SeQuant AB on Saturday, November 29, 2003 - 07:04 am:
¡Hola! No comprende...
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By Anonymous on Friday, December 12, 2003 - 12:08 am:
can you suggest a method for analysing oxazolidinone in hplc
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By Edgar on Thursday, February 12, 2004 - 07:59 am:
I am analyzing strong basic substances with SDS (sodium dodecylsulfonate) as ion-pair-reagent, in an acidic phosphate buffer (pH 3-4) and 5 - 15 % ACN with a standard RP18-column.
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By AllsepTech on Thursday, February 12, 2004 - 08:27 am:
Edgar,
What are you trying to analize? Amines, amidines, aminoacids, quaternary amines? would you provide more details. We have columns with embedded ion-pairing reagent, so you do not have to use SDS-just ACN/water/organic acid (TFA, sulfuric, formic, etc.).
Send us an email with more details.
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By Anonymous on Saturday, March 20, 2004 - 02:01 am:
1 )try with YMC basic 250 x 4.6mm with acetonitrile and ammonium acetate.
2) try with YMC basic 150 x 4.6mm with acetonitrile and ammonium acetate.
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By Anonymous on Tuesday, April 6, 2004 - 02:11 pm:
Question:
SDS adds retention and sharpens up peak shape for amine compound. HPLC conditions are using 30mM/50mM NaPO4/SDS buffer with ACN as a organic modifier. HPLC run under gradient conditions from 48%ACN/52% buffer to 70%ACN/30% buffer. Any suggestions getting reproducible gradient profiles and stable baseline?
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By yader Hernández P. on Wednesday, April 7, 2004 - 02:21 pm:
Se lee bastate interesante tu trabajo, me gustaria que me enviaras mas informacion relacionada con tu trabajo, talves te pueda hacer algunas sugerencia. Yo trabajo en el INstituto de Medicina Legal de Nicaragua con cromatografia liquida y cromatografia de gase con detector de masa. Mi correo es yaderaq@hotmail.com
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By Anonymous on Wednesday, May 12, 2004 - 04:12 am:
Hi,
I am interested in hplc methods for impurities in Digoxin.Are there any safety requirements in handling this compound?
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By Phil on Wednesday, May 12, 2004 - 01:05 pm:
can you guys help me answer some questions about chromatograpy?
Who came up with the idea?
When was the idea first used?
Where is it mostly used today?
Can anything be separtated by using this methood?
What jobs use chromatograpy?
and
How many different types of Chromatography are there?
thanks
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By Anonymous on Wednesday, May 12, 2004 - 01:21 pm:
See the thread "Homework and Test Answers".
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By Russ on Thursday, May 13, 2004 - 05:07 am:
You can also try: http://www.itsjustabox.com
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By Anonymous on Tuesday, June 1, 2004 - 06:16 pm:
Phil,
Make a Google search on "Tswett" and "Chromatography" keywords or read about history of chromatography on
http://www.chromatographyonline.com/lcg ... rticle.pdf
It was invented by a Russian scientist Tswett in 1903. His name "Tswett" on Russian means "Color". People think that he's put his name in the method's name "Chroma"tography. Actually, first chromatographies looked like a set of colored bands (leaf pigments)
------------------------------------------------------------------------------------------------------------
By Anonymous on Saturday, June 5, 2004 - 09:30 am:
Or that he used the colored bands as an excuse to work his name in!
------------------------------------------------------------------------------------------------------------
By Kazimierz on Thursday, July 1, 2004 - 03:18 pm:
Separation of basic compounds:
1. On silica column with nonequous methanol + 0.02-0.1% of HClO4 Flanagan R.J. et al. J. Chromatogr. 247, 15-37, 1982 (amiodarone, DEA)
2. On silica column with methanol:H20 (antipyrine)
3. On Synergy-Polar-RP or Luna C18 (AcCN:H20)2.8%
+ 0.2% HFBA
4. On silica Itertsil: Hydrophilic Interaction LC
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By Kaoula on Friday, July 2, 2004 - 07:08 am:
Hi ALL:
Please i need a clarification about the rule for choice between L7 coulmn and L1 coulmn ??
thank you ver match
Kaoula
------------------------------------------------------------------------------------------------------------
By jack on Wednesday, July 14, 2004 - 07:54 pm:
is anyone have some idea to extract streptomycin in honey matrix and then run on lc/ms/ms, please help
------------------------------------------------------------------------------------------------------------
By Anonymous on Thursday, December 19, 2002 - 09:37 am:
i have a basic compound to run on HPLC, basically the structure is 2 fused rings with primary, secondary, and 3rd-order amine attached or fused into the rings, and a hydroxy group also attached to the ring, the 1st scout run with gradient water/ACN/TFA on C18 column didnot get any good result of course, and the peak didnot retain on column at all. the 2nd try i put an c18-aqua with high percent(98%) water at start, no good; 3rd try i use sodium bicarbonate at pH 8 on the C18 or C18-aqua column, still not retain at all if water decreased to 96% at start.
then i use water/ACN/0.1% TEA with non silica column which is zirchrom hydrocarb column, since the pH with 0.1%TEA is about 11. i still get messy baselines and big broad peak, the sample run can not distinguish from the blank.
anyone have good ideas for a good set of conditions for this?
since the blank run on TEA is also messy, maybe i should try DEA instead?
appreciate any advice.
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By Einar Pontén - SeQuant AB on Thursday, December 19, 2002 - 11:14 am:
I suggest that you try HILIC mode of separation.
The ZICâ„¢-HILIC column is well suited for basic compounds.
http://www.sequant.com
------------------------------------------------------------------------------------------------------------
By Chris Pohl on Thursday, December 19, 2002 - 12:13 pm:
I would suggest trying cation exchange. Since you are reporting insufficient retention via reversed phase even in the presence of high water content eluents, the sensible strategy would be to accomplish retention based on the analyte's fundamental property: its cationic nature. Most likely separation can be accomplished using any of the many available silica or polymer based materials, although the tendency of your analyte to strongly interact with silanols may compromise chromatographic performance on silica based materials. Alternatively, one can use any of a number of available polymer materials. One material specifically designed for such analytes is the PCX-100 column (http://www.dionex.com/app/tree.taf?asset_id=9324). Best performance is generally achieved for analytes of this sort with potassium or better still cesium based eluent systems containing 20% acetonitrile.
------------------------------------------------------------------------------------------------------------
By Daren on Thursday, December 19, 2002 - 01:09 pm:
I also think HILIC is good idea, but if you are looking to stay with reverse phase you might want to try a more polar stationary phase. A good choice for your application might be the Waters Xterra Phenyl column. You can run it in 100% aqueous if needed. I would stick with TFA and keep the pH low (around 2),you may get added retention from ion-pairing interaction of TFA.
Since you have so many amine functional groups with varying pKa your going to have trouble getting a sharp peak once you start raising the pH.
Daren
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By Daren on Thursday, December 19, 2002 - 01:13 pm:
you can always throw in an ion-pairing agent at low pH like octane sulfonic acid and go back to the C18. But I tend to find ion-pairing methods not very robust.
Daren
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By Einar Pontén - SeQuant AB on Thursday, December 19, 2002 - 01:28 pm:
Beside the separation problem I think you should also consider what different detection techniques you may need today and later on.
If MS, then ion-pairing and traditional ion-exchange may be less suitable.
------------------------------------------------------------------------------------------------------------
By Uwe Neue on Thursday, December 19, 2002 - 03:52 pm:
There are several possibilities to solve the problem. First, you can use one of the columns that have been optimized to give good retention in 100% (!!!) water (do not use 98% or 96% as you did). A column that might be useful for you is the Atlantis d-C18 column from Waters that has been designed for this kind of difficulties.
You can also go to a higher pH, but I would not use a carbon column. You can use one of the newer C18 zirconia columns, or an XTerra MS C18 with ammonia or ammoniumbicarbonate at pH 10 as a mobile phase. You can also change the retention pattern completely, and use hydrophilic interaction chromatography combined with ion exchange. In order to do this, you should use a silica column. They are nicely retentive, and they do not give tailing peaks, since there is no bonded phase to hinder the interaction with the silanols. Considering the description of your compound, I believe that the last approach will work best: you will have plenty of retention, you just need to get used to how to manipulate retention in this mode of chromatography. I can get you some advice, if needed.
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By Michael Goodwin on Friday, January 10, 2003 - 03:04 pm:
I have a similar situation and thus posting it here instead of as a new question.
I am developing a method for quantitation of a small peptide (~650Da) that has 4 amine groups. The compound is provided in a disulfate salt form.
On reverse phase columns (beta basic C8 Hypersil, C18 phenomenex luna2, Discovery F5) the compound is unretained in all mixtures of water/acetonitrile each w/ 0.1% v/v formic acid.
The compound is added to 0.1M KOH to attempt desulfating and return to free base form. Still no retention.
On Silica, only high levels of water:MEOH elute compound, and does so quickly. When compound is added to mouse serum matrix, the matrix co-elutes and interferes with detection (DAD at 269nm, also have ES-MSD available)
I have switched to a PRP-X200 SCX 2.1mm x 15cm 10u particle column that has low capacity exchange sites.
mobile phase is:
A = Water w/ 0.1% v/v TFA
B = solvent A w/ 500mM NaCl
C = Acetonitrile
flow = 0.5 ml/min
With the TFA and NaCl, I have stopped adding the peptide sulfate into KOH and just inject it as is.
I am able to retain the compound and elute it, however I am experiencing a lot of peak broadening.
Some examples include:
mobile phase A:B:C = 45:30:25 k'=18.9 w/ 50% peak ht width (pkw)= 3.1 minutes
A:B:C = 30:45:25 k'=3.8 pkw = 1.8 min
A:B:C = 0:75:25 k'=0.67 pkw = 0.2min
A:B:C = 40:30:30 k'=3.32 pkw = 2 min
A:B:C = 20:30:50 k'=1.5 pkw = 1.3min
Essentially, if I increase acetonitrile concentration or salt concentration the peak gets narrower, but retention suffers greatly which will increases the chance that matrix components will coelute. Peak shape only looks good when compound is essentially unretained.
Decreasing organic or salt gives better retention with unacceptable peak broadening.
test sample concentration is 10uL injection of ~0.5ppt compound. 0.05ppt concentration does not improve peak broadening so I do not believe I'm saturating the exchange sites. The problem persist with 0.2 and 1 ml/min flow rates.
Any ideas on how to get retention with narrow peak width?
I'm thinking next to try the above suggesstion of purchasing octane sulfonic acid to take advantage of the RP character of the PRP-X200 column.
Or, are the amine groups binding to silanols on the PRP column and perhaps it needs some end capping agent??
Any suggestions or explanations will be greatly appreciated.
Thank you.
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By Uwe Neue on Friday, January 10, 2003 - 04:05 pm:
You have an interesting problem.
The PRP column is not based on silica, and therefore does not have silanols. The broad peaks are most likely due to the fact that you are using a polymeric packing. These packings tend to give broader peaks.
If I understand this correctly, you ultimately want to determine your compound in a mouse serum matrix. Let me propose to you an approach that is similar to the ideas that Naidong Weng has published, and that I am currently excited about. I believe it was published in the Rapid Communications in MS, if I remember correctly.
Let us first worry about the HPLC separation, then let us discuss the sample preparation. I recommend to use a silica column in HILIC/ion-exchange mode. You have had very good success with your standard. Your compound is retained on silica in methanol-water. You can manipulate this retention by changing the methanol concentration (more methanol = more retention) or with pH (silica is a weak cation exchanger). You should end up with a mobile phase with a fairly high methanol content.
For the sample preparation, I would now try to do an acetonitrile precipitation first. Dilute your serum sample 5:1 with MeCN! This precipitates the proteins, and you can inject the supernatant directly into your HILIC HPLC. If the stuff is still too dirty, you need to do SPE for sample clean-up. I would use a cation exchange method. Load your sample onto an Oasis MCX SPE device, wash with water/acid, then with methanol/acid, then elute with a high concentration of triethylamine in methanol. You can find details of the procedure on the Waters web side, but you may need to increase the concentration of base to get your sample off (I expect it to be strongly retained on the ion-exchanger since you said it has 4 amino groups).
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By Uwe Neue on Saturday, January 11, 2003 - 04:03 pm:
The Weng Naidong paper is in Rapid Communications in MS 2002; 16; 1965-1975
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By Michael Goodwin on Monday, January 13, 2003 - 10:43 am:
Thank you Uwe. I'll try out the suggestions and post any progress.
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By Einar Pontén - SeQuant AB on Monday, January 13, 2003 - 03:14 pm:
The small and basic tripeptide - Gly-His-Lys - was retained on a ZICâ„¢-HILIC column (high conc acetonitrile). This tripeptide showed more tailing than the basic polypeptides;
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe and
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg.
When the ion strength was increased the tailing for Gly-His-Lys decreased, while k' only changed from 5.8 to 4.6. The polypeptides (k' 2.2 and 3.4) were less affected.
Thus, in this case the zwitterionic functional group showed a selectivity based on both charge and hydrophilicity. The ion exchange mechanism was suppressed by a higher ion strength in the mobile phase.
I suppose that your "small peptide (~650Da) that has 4 amine groups." would behave as the polypetides mentioned above.
I agree with the sample preparation procedure suggested by Uwe Neue. If too high concentrations of the amine is required, then a ZICâ„¢-HILIC SPE could be an alternative.
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By Chris Pohl on Tuesday, January 14, 2003 - 12:14 pm:
Michael Goodwin,
Regarding your original question, a point to keep in mind is that the nature of your eluting cation is an important consideration in terms of obtaining good peak shape with some polymeric stationary phases. I'm not sure exactly how PRP-X200 is sulfonated but it's likely that this has produced some relatively sluggish mass transport. Potassium based eluent systems typically significantly improve chromatographic performance under these conditions and cesium based eluent systems will work even better.
In addition, contrary to the comments above by Uwe, modern polymeric stationary phases are commonly comparable in chromatographic efficiency to the silica based materials of the equivalent particle size with the added advantage of much better hydrolytic stability. Another option worth considering is a switch to a pelicular cation exchange material such as the PCX100, available from Dionex. This material provides excellent peak shape for a variety of cationic analytes which prove problematic on silica based materials.
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By Anonymous on Wednesday, January 15, 2003 - 10:27 am:
Chris,
what do you mean with cesium based eluent systems?
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By Chris Pohl on Wednesday, January 15, 2003 - 03:44 pm:
Anon above,
Cesium based eluent systems provide superior chromatographic efficiency in polymeric cation exchange systems. In the example above, the dominant eluent cation was sodium (from the sodium chloride). Substituting cesium chloride under the same conditions will produce significantly better performance. Keep in mind, since cesium is a more potent eluent cation, it will be necessary to adjust the concentration of the cesium accordingly.
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By Uwe Neue on Wednesday, January 15, 2003 - 04:15 pm:
Chris,
I have little experience with ion exchange, but we have looked at most modern reversed-phase columns, including polymeric packings. None of the porous polymeric ones had mass transfer coefficients that reached a porous silica-based packing. Of course, you can argue that non-porous packings have a better mass transfer coefficient, but due to the compromises involved in these packings, none of them is anywhere close to where they should be.
Close, but no cigar!
Best regards
Uwe
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By Benjamin on Thursday, January 16, 2003 - 06:04 am:
Chris, Uwe;
Over 20 years I have tried to use polymer-based columns of all types. Everytime I develop a new method or separation I always consider their use, many times I have tried them, and almost invariably, I end up going back to silica ones.
I think it is fair to say that polymer ones just do not have the efficiency we would like to see. I have yet to obtain a decent reversed-phase application using them. However, I have obtained some use from them in ion-exclusion and ion exchange.
Benjamin
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By Chris Pohl on Thursday, January 16, 2003 - 09:26 am:
Uwe, Benjamin,
First of all, I'm afraid this discussion has devolved into personal opinion dominating over relevant factors. The fact is, in my 30 years of experience in HPLC and Ion Chromatography I've had a chance to use a lot of different materials for separation of ions. Whether or not a polymeric material has comparable efficiencies, higher efficiencies or lower efficiencies depends upon the analyte and design of the stationary phase. It is not useful to generalize about the relative merits of polymeric and silica based phases based on generalizations surrounding efficiency. The fact is that selectivity is the dominating factor in stationary phase design and in my experience polymeric phases afford much more selectivity control than silica based materials. It really doesn't do any good if a particular column has more theoretical plates than another if the job at hand requires, say resolution of bromate and chloride, and the two analytes coelute on the column with the highest efficiency. Furthermore, polymeric columns are typically considerably more stable. A silica column that last six months is doing well but I have seen data on polymeric columns that have been in use for five years at pH 13 with no significant degradation. Perhaps, it's all just a matter of personal opinion. The preceeding probably will not convince you. So, can we just agree to disagree?
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By Benjamin on Thursday, January 16, 2003 - 11:25 am:
Chris;
I agree to disagree to agree (just kidding). Basically I feel that your comments agree with mine. Polymeric materials are more adequate for the analysis of ions (ion exclusion, ion exchange). But as Uwe says and I mentioned, the same type of materials do not perform as well in reversed-phase separations. Even if silica-based columns are not very long-lasting, they are still the best option in many cases.
I believe you have polymeric materials that have been used for 13 years or more. Please also believe me that I have some, older than 10 years, and I have not been able to obtain any good results from them.
Benjamin
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By Michael Goodwin on Thursday, January 23, 2003 - 02:07 pm:
Still have some work to do with these suggestions, but I figured I should chime in and let everyone know that I'm still around and listening.
I'll post more details when the study is complete, but here's the short of it.
Switched to HILIC using ammonium formate/NaCl and Methanol and had difficulty eluting the compound off the column.
Briefly switched to PRP-X200 using CsCl instead of NaCl.
Compound eluted quicker, but still alot of peak broadening.
Switched to HILIC again with CsCl and seeing promising results. I have a good feeling about this combo and adjusting CsCl in the mobile phase may give the right optimization. More to come.
I also found the discussion of polymer vs. silica interesting.
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By Einar Pontén - SeQuant AB on Thursday, January 23, 2003 - 02:17 pm:
Yes, the agree-to-disagree dialog is intereting.
Michael,
Why don't you send us a sample and have the problem solved....?
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By Anonymous on Friday, January 24, 2003 - 07:41 am:
What is more soluble in MeCN? CsCl or NaCl?
Thanks
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By Chris Pohl on Monday, January 27, 2003 - 01:03 pm:
CsCl is considerably more soluble in organic solvents than NaCl although in both cases the solubility is pretty low unless some water is present.
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By Michael on Friday, May 23, 2003 - 12:34 pm:
This has been concluded for awhile, but I figure better late than never. The final method for these extremely polar peptides:
Silica column 5u 4.6mm * 250mm in HILIC mode:
mobile phase: 75% (.1%v/v acetic acid with 3mM CsCl) and 25% v/v MeOH isocratic flow 1.0 ml/min
Preparation: 1:1:5:13 serum:internal std (aq w/ acetic acid):water with .1% acetic acid: acetonitrile (added last)
acetonitrile ppt out serum components and leaves enough internal standard and sample for quantitation with uv detector at 269nm. samples are also centrifuged 5 min at 4000RPM to reduce turbidity.
CsCl and acetic acid worked wonders over NaCl.
peptide of interest elutes with baseline resolution as two peaks (can now resolve both components) with k'~ 0.9 & 1.0 peak width ~0.2min. Internal standard (same structure, one less hydroxyl group), also possesses baseline resolution. All three components have great peak symmetry with no obvious tailing.
Serum peaks virtually non-existent and elute in void volume.
I truly appreciate all the suggestions provided.
thank you
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By CHVS NAGARAJU on Wednesday, July 30, 2003 - 05:31 am:
What is more soluble in MeCN? CsCl or NaCl?
Solubility is depend on the polarity of the solute and solvent. Based on 'Like solves like' concept Cesium chloride is more soluble than Sodium chloride in Acetonitrile(CH3CN).
As per the elotropic series(Silica based), n-hexane is having zero polarity where as water maximum.
Acetonitrile's polarity is 5.8
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By Andreas Neumaier on Friday, August 1, 2003 - 07:48 am:
To get back to Anon who started the tread:
You are using TFA with a basic compound so you are getting an very polar ion pair which don't retain (much) on typical C18. The lower the pH of the mobile phase the lower the retention time.
When you use a fluorinated phase (Fluofix, Fluorophase RP or Fluosil or any other column aviable) the TFA-ion pair will be retained quiet good on the fluorinated phase (even pure TFA is retained). And (from my experience) the funny part of it is: basic compounds with lots of basic N "bounds" more TFA and is retained stronger on a fluorinated column.
With other words: Using a fluorinated phase and TFA with a compound (which is able to build TFA-complexes or TFA-ion pairs) is one of the simplest ion pair systems and the mobile phase is very easy to mix (just add an exactly volume of TFA to the mobile phase).
I have run into some problems with fluorinated phases (some of them happens with ALL silica columns):
- Never let TFA inside the column when it isn't used. Flush the column every time it won't be used for some time (hours).
- Tailing with basic compounds can be connected to the concentration of TFA in the mobile phase. Double the amount of TFA and check tailing again.
- Keep an eye on the pH of the mobile phase. It is possible to operate even at low pH (2 to 2.5) for some time when the column is flushed after the last injection.
- Check the UV absorbance of the mobile phase when operation at low wavelengt. TFA is strongly absorbing at low wavelenght (bye bye linearity...).
- When ordering a fluorinated phase, take at least two different batches. If the columns differs too much, send them back, take your money and change to an other manufacturer.
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By Anonymous on Thursday, August 21, 2003 - 11:41 am:
Hi everyone
Please suggest HPLC conditions for the determination of cyclosporin A related substances.
Thanks
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By Anonymous on Friday, November 28, 2003 - 08:46 pm:
Soy estudiante de maestrÃa en ciencias animales, quiero evaluar los componentes de la fracción etanólica de hojas de Ambrosia cumanensis, por medio de HPLC. Qué me recomiendan para tener en cuenta en el procedimiento?
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By Einar Pontén - SeQuant AB on Saturday, November 29, 2003 - 07:04 am:
¡Hola! No comprende...
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By Anonymous on Friday, December 12, 2003 - 12:08 am:
can you suggest a method for analysing oxazolidinone in hplc
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By Edgar on Thursday, February 12, 2004 - 07:59 am:
I am analyzing strong basic substances with SDS (sodium dodecylsulfonate) as ion-pair-reagent, in an acidic phosphate buffer (pH 3-4) and 5 - 15 % ACN with a standard RP18-column.
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By AllsepTech on Thursday, February 12, 2004 - 08:27 am:
Edgar,
What are you trying to analize? Amines, amidines, aminoacids, quaternary amines? would you provide more details. We have columns with embedded ion-pairing reagent, so you do not have to use SDS-just ACN/water/organic acid (TFA, sulfuric, formic, etc.).
Send us an email with more details.
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By Anonymous on Saturday, March 20, 2004 - 02:01 am:
1 )try with YMC basic 250 x 4.6mm with acetonitrile and ammonium acetate.
2) try with YMC basic 150 x 4.6mm with acetonitrile and ammonium acetate.
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By Anonymous on Tuesday, April 6, 2004 - 02:11 pm:
Question:
SDS adds retention and sharpens up peak shape for amine compound. HPLC conditions are using 30mM/50mM NaPO4/SDS buffer with ACN as a organic modifier. HPLC run under gradient conditions from 48%ACN/52% buffer to 70%ACN/30% buffer. Any suggestions getting reproducible gradient profiles and stable baseline?
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By yader Hernández P. on Wednesday, April 7, 2004 - 02:21 pm:
Se lee bastate interesante tu trabajo, me gustaria que me enviaras mas informacion relacionada con tu trabajo, talves te pueda hacer algunas sugerencia. Yo trabajo en el INstituto de Medicina Legal de Nicaragua con cromatografia liquida y cromatografia de gase con detector de masa. Mi correo es yaderaq@hotmail.com
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By Anonymous on Wednesday, May 12, 2004 - 04:12 am:
Hi,
I am interested in hplc methods for impurities in Digoxin.Are there any safety requirements in handling this compound?
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By Phil on Wednesday, May 12, 2004 - 01:05 pm:
can you guys help me answer some questions about chromatograpy?
Who came up with the idea?
When was the idea first used?
Where is it mostly used today?
Can anything be separtated by using this methood?
What jobs use chromatograpy?
and
How many different types of Chromatography are there?
thanks
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By Anonymous on Wednesday, May 12, 2004 - 01:21 pm:
See the thread "Homework and Test Answers".
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By Russ on Thursday, May 13, 2004 - 05:07 am:
You can also try: http://www.itsjustabox.com
