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Chromatographic Method Development - Science topic

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I am developing a gradient method for the estimation of 3 drugs, two are water-soluble and one is hydrophobic. I am using Dionex HPLC system equipped with P680 HPLC pump, ASI-100 automated sample injector, UVD340U detector. The mobile phase is composed of 10mM KH2PO4 buffer pH 6.8 and ACN. A gradient is applied from 97:3 (or 90:10, depending upon the column used) Buffer: ACN up to 5 mins, followed by 50:50 buffer: ACN up to 16 mins, followed by 97:3 Buffer: ACN up to 20 mins. Columns tried are Inertsil ODS 3, Inertsil ODS 3V, Eclipse XDB c18, Phenomenex Gemini C18. Samples prepared in 50:50 Water: Methanol.
After 30-50 injections, the pressure rises to 170-200 bars (initial pressure 96-106 bars). After back flushing or washing at 50 deg celsius using 60:40 ACN water, the pressure reduces. However, again with increasing the aqueous content beyond 60%, the pressure starts rising and the peak shape is also not good. What could be the reason?
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In my opinion try to use Methanol instead of Acetonitrile.....
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I am having baseline issue when following the method outlined in a application note from Agilent. The application note references a method that utilizes a gradient in the attached image.
The whole application note can be found by searching for 5994-3791EN.
I do have a binary pump instead of the quaternary pump, but otherwise there is very little difference between my instrument's config and the reference method that would significantly effect afaik. If Agilent's newer software corrects for the baseline issues seen maybe that could be the answer.
I also have the older Agilent DAD from the 1100 series if that is relevant.
What could possibly be the root cause of the difference in baseline seen here?
The transition dip is seen at other gradients of these same two mobile phases.
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My study involves establishing chronic nicotine addiction in mice and I would to check the cotinine level in mice urine using HPLC but currently protocols I found are done using human urine. Bioassays and GC are expensive, and I would like to use urine as the sample. Thus, how do I modify the HPLC protocol for mice urine?
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I have no practical experience, however, I hope the provided link will be very much helpful for you. Please have a look on the following link:
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I am using HPLC under the directions of 0.6ml/min 0.01M H3PO4 solution, 40 degree to test methyl formate and formic acid together. However, the peaks for both appear around the similar time so they overlap. I want to know how to change the conditions to let the two peaks separate.
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I used HPX-87H column to seperate methyl formate and formic acid, but failed.Does anyone have any advice?
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I want to purify such kind of 2,6-Bis(3-methylimidazol-1-ylidene)pyridine dibromide or 2PF6 salts via column chromatography. These dibromide salts are soluble in water and PF6 salt are soluble in acetonitrile. In this solubility condition which kinds of eluent, should perfectly work for column chromatography...?
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Hi, would you help me to prepare
1,1′-(2,6-Pyridinediyl)bis(3-methylimidazolium) Dibromide, I couldnt find any good paper. Thank you
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Hi Research gate community,
I'm currently working on HPLC/Electrochemical detection method development for a peptide Hepcidin-25 (also known as iron regulator). After trying different gradient profiles with phosphate and acetate buffers in acetonitrile and methanol respectively, I came to the conclusion that Hepcidin-25 is not electrochemically active.
So I began searching about techniques that can be used to make amino acids and peptides electrochemical active and found that the most popular technique is derivatizing with OPA/ beta-mercaptoethanol or OPA/sulfite, for aminoacids. Although this technique was never applied to peptides, I'm willing to try and check if some amino acids in the peptide are going to be derivatized. Biuret's method was found to be successful for peptides, but it was never used with HPLC and required a very high potential to oxidized for some peptides.
Does anyone know if there are any other methods that may be worth exploring to make this peptide electroactive? or otherwise, I have no option but to collaborate with labs equipped with HPLC-MS/MS.
Thanks in advance!
Amino acid sequence of Human Hepcidin 25 is:
Asp-thr-his-phe-pro-ile-cys-ile-phe-cys-cys-gly-cys-cys-his-arg-ser-lys-cys-gly-met-cys-cys-lys-thr
Mobile phases combinations tried:
(A: 50 mM NaH2PO4 (pH~2) in water, B: 100mM NaH2PO4/ acetonitrile/methanol 30/60/10 (v/v/v) pH~2) and
(A: 100 mM Lithium acetate in water, B: 100 mM Lithium acetate/ methanol/ acetonitrile 10/80/10 (v/v/v))
Column: C18, 4.6 mm x 250 mm, 5 um, 300A
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Hello,
There are a few different ways to approach your problem.
With 8 Cys present, hepcidin-25 should be electrochemically active. It may be that your sample is oxidized and no longer detectable. The easiest way to address this is to treat the samples (standards and biological0, first with a reducing reagent like dithiothreitol (DTT). Selvi shows how to do this with glutathione.
(Selvi, M. Bharath. "Biochemical Mechanisms of Homocysteine and its Role in RETINAL VASCULAR DISEASES." PhD diss., BITS Pilani, 2015.
See also: Sakhi, A.K., Russnes, K.M., Smeland, S., Blomhoff, R. and Gundersen, T.E., 2006. Simultaneous quantification of reduced and oxidized glutathione in plasma using a two-dimensional chromatographic system with parallel porous graphitized carbon columns coupled with fluorescence and coulometric electrochemical detection. Journal of Chromatography A, 1104(1-2), pp.179-189.
Zhang, M. and Pfeiffer, C.M., 2004. Comparing the ESA HPLC total homocysteine assay with electrochemical detection to the CDC in-house HPLC assay with fluorescence detection. Clinica chimica acta, 340(1-2), pp.195-200.}
Standards of glutathione or oxidized glutathione (10 µg/mL) are made in buffer. 50 µL of standard is mixed with 5 µL of 50 mM DTT at room temperature for 10 minutes. 10 µL of the mixture is injected into an HPLC (Buffer: 0.1% trifluoroacetic acid, 2% acetonitrile, 98% MilliQ water; Flow rate: 1 mL/min; Pressure: 400 bar; Temperature: 27.7 °C; ECD with glassy carbon electrode; Range: 1 µA; E cell: 0.90; Voltage: 1.16V; Column: ODS Hypersil C-18, 5 µm particle size, 150 X 4.6 mm).
Note that the moles of disulfide bonds per gram of oxidized GSSG is different than the moles of disulfide bonds (possible) in hepcidin-25 by ~1.75:1. So you might want to make the DTT solution a little stronger (~90 mM). You could also add more 50 mM DTT solution but that will dilute your sample.
Since you are open to other methods, I have attached a chapter on thiol-reactive fluorescent dyes that you can use to label Hepcidin-25. Again, remember the stoichiometry and be sure to first reduce any disulfide bonds in Hepcidin-25 (intra- or inter=molecular) and also use enough thiol-reactive dye to label all 8 Cys.
Best regards,
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In my HPLC method development, Active peak cumming negative in sample and normal in standard solution. Can anybody suggest the reason behind this?
Instrument method:
Mobile phase : 1% Aceetic acid in water and Acetonitrile 52:48 % v/v.
Diluent : Methanol
Column: Zorbax Eclipse Pluse
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The method was invalid as shown. The consequences of using instrumentation and techniques without proper training often lead to erroneous data being collected and incorrect conclusions or quantitative results being reported. In your case, there are two reasons for this:
(1) The DAD reference wavelength feature on your Agilent 1260 DAD was turned 'ON'. This must be turned 'OFF', not 'ON'. To learn why, please read the attached article below.
(2) Your signal acquisition wavelength is very close to your Reference Wavelength. This guarantees that your data will be invalid as even the peak of interest will fall under the Reference range you specified (360,10), invalidating the method and data obtained.
Those who use HPLC for the first time and/or who have not had any formal training in liquid chromatography (and especially in the use of a diode array detector (aka: PDA)) often makes these mistakes. Clients often use systems setup just like this one and are unaware of the problems caused until a negative peak shows up. The appearance of the new, negative peak is this hint that all of your previous data may be invalid and the only reason you discovered it now is because this time the system had to subtract-out so much data from your main peak that it ended up being negative!
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We are looking for a reliable method or procedure to identify all biological compounds in Cell Free Supernatant (CFS) of lactic acid bacteria.
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Added an answer
Goodluck
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I have been trying to develop a TLC system for an unknown compound (biosurfactant), but there are so many spots and trails in TLC and no solvent and their ratios have been proven appropriate for their characterization. I want to purify the compound by TLC cuttings so I need to develop a solvent system which is best suited for the separation of all the components present. Will you all please suggest some combinations of solvents which are universal for all compounds?
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I have face same problem, but in my situation i have a purified compound using column, that is soluble in methanol. For estimation of its quality using tlc plate which solvent system is best.
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I read in Mass spec literature that buffer combinations are attempted. Are they only for gradient elution. Does this need mass spec to ionise the buffered solvent effectively. Will this cause the buffer to precipitate. 
how does one make up such a buffer solution so simply?. Should I add formic acid to the ammonium acetate + ACN mixture already or add formic acid to 10ml of filtered ammonium acetate?. 
Reverse phase is highly organic mobile phase at 90-97% ACN.
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Yes you may use salt for several reasons.
See why and how to prepare in the attached HILIC tips
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Anal Chim Acta. 2017 May 15;967:12-32. doi: 10.1016/j.aca.2017.01.060. Epub 2017 Feb 7.
Recent advances in stationary phases and understanding of retention in hydrophilic interaction chromatography. A review.
Jandera P1, Janás P2.
I was looking for efficiency of gaurd column on its own for various compounds and for linking up of gaurd columns.
The fact is that e.g. phenyl column coupled to C18 column improved resolution (related to void volume in capacity factor) defied my understanding of  mixed mode chromatography.
The journal: J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Dec 15;941:116-22. doi: 10.1016/j.jchromb.2013.10.005. Epub 2013 Oct 16.
Liquid chromatographic mass spectrometric method for simultaneous determination of smallorganic acids potentially contributing to acidosis in severe malaria.
Sriboonvorakul N1, Leepipatpiboon N, Dondorp AM, Pouplin T, White NJ, Tarning J, Lindegardh N.
serially links ZIC HILIC Gaurd column to ZIC HILIC which is common research for polar analytes. 
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I have done the flash column chromatography of the crude mixture. After the slow elution at 4% Ethyl acetate in petroleum ether, the polar impurity is separated and there is not signal of the impurity in flash chromatography. when the compound elution starts, the initial non-polar impurity still persists and comes along with the final product. I have tried washing with n-hexane and methanol. The final compound and impurity also goes into the non-polar solvent layer, and methanol layer also have both the compounds. I dont know how to separate this impurity from the final compound. Rf of impurity is 0.98 and Rf of compound is 0.65.
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I'm assuming you are using the same elution for the flash as is being used for the TLC. The Rf shown doesn't  suggest the chromatography will be good. The non-polar compound has an Rf of (essentially) 1, and the product has an Rf of 0.65. Retention on a column is, in column volumes (CV)= 1/Rf. The column volume approximates the void volume on an HPLC. So the impurity comes out at 1 CV and the impurity comes out at ~1.5 CV. The column volume is merely the space taken by the mobile phase in the column and nothing can elute faster than 1 CV.
You would prefer the compound to come out at 2-5 CV, or an Rf of 0.2 through 0.5 and that will be difficult to achieve as you are running 4% EtOAc in pet ether now . I suggest trying a less polar mobile phase such pet ether/THF or pet ether/ether. One can also try pet ether/toluene. Pet ether/dichloromethane (DCM) could work but DCM is nearly as polar as EtOAc. Likewise, pet ether/acetone is similar in polarity to pet ether/EtOAc and so probably won't work well.
For TLC, make sure the chamber has time to saturate with vapor, and make sure vapor can't escape. A watch glass covering a beaker doesn't give the best results because the beaker spout allows vapor to escape. Use a proper TLC chamber or jar that can be completely closed.
If you are running a flash column on an instrument with such a solvent system, make sure the equilibration is aet to 5-7 column volumes. The EtOAc adsorbs on the silica, and compounds often don't elute until this adsorption is complete. If packing columns, pack in at least 1-2% ethyl acetate of you are running a step gradient for the same reason. People often dry-pack columns and this may be one case where that can adversely affect the chromatography.
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Hi. I am developing an analytical method based on the quantification of mofetil mycophenolate (MMP) and mycophenolic acid (MPA). For this purpose HPLC with a reversed phase stionary phase column (ODS-C18) and a mobile phase based on a typical phosphate buffer solution and acetonitrile, with a high column temperature, is being used. The literature describe some methods were retention times for MPA are always lower than those for MMF. In my method is just different. Firstly appears MMF (Rt - 7.00) and then MPA (Rt - 16.0). Any Idea for this? Why elution order is different for both molecules if I am using same separation mode based on same stationary and mobile phases? Thx.
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Dear Carlos,
how did you confirm the identity of the two drugs? Dou you analyze standard solutions of both drugs separately? I mean, are you sure the identity of the two peaks obtained?
Regrds.
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I am using reversed phase columns HP-5 in GC and C-18 in HPLC
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MOBILE PHASE A:buffer NH4HCO3 10 mM ? PH 10 : acetonitrile 60%:35%
MOBILE PHASE B: ACETONITRILE
FLOW RATE:1ml/min
colum temp :35°c
gradient composition :
phase                             time                      A%                                    B%
1                                     0.01'                    100                                      0
2                                       5'                        100                                      0
3                                       35'                         5                                      95
4                                      36'                           5                                     95
5                                        40'                            100                                 0
run time:40'
post time 10'
i am surching for impurties of 1-, pipéridine-4-carboxamide
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If you are going to use the column in the near future just flow 10X column volumes of Mobile A and B, without the buffer.  It really depends on your sample matrix and the crap that is injected.  Long term storage is given by the manufacturer.
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I need to detect styrene monomer from different food sample
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Dear Naziruddin
As said by Mr. Neshat, you can use the same calibration run for each sample. You can add another internal standard inside you sample if you want to see the stability of the sample too. But for the same compound, you can definitely use the same calibration run, with the same method.
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My compound is Di ethyl phthalates
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No HPLC method information was provided. That means there are hundreds of reasons why it might happen. It is usually something very simple. As Jack suggested, wash the column (properly with a stronger solution, NOT the mobile phase which will do nothing at all) OR replace the column with a NEW one and see what happens.
Additionally, please review this article for ideas?
  • "HPLC Peak Splitting. Common Reasons For It"
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I am using an anion exchange column for my experiment and store it in 20% Ethanol as recommended. When the column is put to use weekly it gives a proper baseline with minimum stabilization.
However, when used after a period of 15 days or more, it gives a very wavy baseline and low resolution. 
Any suggestions on alternative storage solutions that can be used or storage techniques that could be implemented?
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I have never stored a ion exchange column in anything other than the eluent I was running it with.
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I am using an Agilent GC MS
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Do you work in SIM or SCAN mode? In case you are working in SCAN mode, what is m/z rage you are using? Can you identify (based on MS spectra) the compound giving you the problem?
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Separation of epoxides using a B-bex column with acetonitrile as solvent and deterctor FID. The conditions are: oven temperature-max 70 ° C, injector 280 ° C, detector 220 ° C, pressure 38 psi. I used the method around 30 injections, all normal, when finishing one of them, the baseline jumps and generates a lot of noise, from there, in all the chromatograms the baseline shows the temperature ramp that I use. What happened to the column?
Blue line - normal chromatogram. Green line - first time it appears.
Purple line and aquamarine - chromatograms after the jump
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First off, do you mean a b-DEX column? That is a chiral phase, and has to be handled with great care. 
You claim to be running an analysis in acetonitrile but you show that your maximum temperature is well below the boiling point of the acetonitrile. As is obvious to anyone with any experience in chromatography this would simply not be possible. If this is the actual case then you need to completely change your analysis. If you truly are using ACN as your solvent (a very poor choice for GC, by the way) then you are building up solvent all through your system and you are probably flooding your detector. This is consistent with the chromatographic traces that you have shown.
Secondly, 220C for a FID is WAY too low. You will get all kinds of crazy things happen. Raise your detector temperature up to no less than your injection temperature. 280 C as an injection temperature for a maximum oven temperature of 70 C is WAY, WAY too high!!! Lower your injection temperature.
You are in a university environment. You clearly are in need of some very basic GC training. Surely your university has someone who actually knows something about GC? You should consult them.
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I am working on biosurfactant production  and one problem is lease help me as When I perform HPLC on mixture as Sigma standard I see more than one peak. The same is on HPLC in other published results with isolated rhamnolipids.
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This is not unusual!  Even a 'standard' that is 95.0% 'pure' has 5.0% other stuff (crap, process intermediate)'.  Thus, you are likely to have many peaks.  In addition a standard that is a DL racemic mixture would have 50% D and 50% L stereoisomer.
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I inherited a poorly working method for a 93kDa protein that needs significant revision.  Column is a waters XBridge BEH C4 300A and mobile phase is 0.1% triethylamine in water (A) and ACN (B); diluent is 1% SDS (also confusing me).  During development when mobile phase was modified with TFA or formic acid it lead to significant and persistent carryover. When TEA   was tried (~pH 12) the carryover got better to be workable but when i titrated the TEA with acetic acid for TEAA  to get the pH to a more reasonable operating range the separation fell apart; at 0.1% TEA i am burning through columns rapidly as i am at the operating limit.  Why could this be happening; it seems more dependent on the pH than the ion-pairing reagent?  Also the protein is in an oil-in-water emulsion and a liquid/liquid extraction is used to isolate the protein and remove the vehicle, is there any other methods to be used to possibly dilute and shoot? any guidance on approach from the community is greatly appreciated!
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very strange doesn't begin to explain this protein and the protein has no native environment; it is an engineered hodgepodge of several antigens expressed as a single protein.
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I am doing method development. on one run of 100 ml of mobile phase pressure is 190kgf, second run of 100 ml of measuring cylinder of mobile phase pressure is 150 kgf, third run 120 kgf. This cylces repears as such. Same is the case with retention time sometimes 5, sometimes 5.5 sometimes 4.3.
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Great answers given above. I would add one thing:  Can you watch the column pressure in real time?  Is it a gradual change in pressure or erratic? If it's erratic, it could be a seal or air bubble.  Make sure the lines are primed well when you switch mobile phases. 
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I treid to rectify it by using 2ml/min flow rate through the coloumn for atleast 5 hrs. Even now when I start the equipment pressure is not stabilising.
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First, terminology. It is all called "HPLC".  Terms like "UFLC" and "UPLC" are trademarks ("UFLC" https://trademarks.justia.com/772/99/prominence-77299484.html & http://hplctips.blogspot.com/2015/08/terminiology-which-is-it-uplc-uhplc-or.html).
Please refer to this free, linked article to solve your problem, "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline) ". I am sure you will find the solution after following the suggestions provided in the article.
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I am facing the problem that the chromatogram of Methomyl is not beautiful!. As you see in the attaching picture below. there are some peripheral peaks beside the main peak, that is the best result I have got until now. I use Methanol and formic acid 0.1% as mobile phase with Gradient pumping method. I suppose that Methomyl was degraded  but in many paper, they said methomyl is stable in acidic medium. Can someone tell me the reason and recommend the suitable solution.?
thanks all
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More information is needed. Please provide the detailed chromatography conditions (flow rate, exact mobile phase composition and times, injection volume, plus ALL HPLC column dimensions and type).
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We would be interested in 50mm column diameter scale. We are looking at Waters, Agilent, Shimadzu. Any comments on robustness? Customer support? Bad experiences?
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Hi Dean,
there was a similar question about a year ago on RG, I attached the link FYI. In general, every company will tell you that they are the best. We use Agilent 1200 system. However, we never had a bad experience regarding the customer support. From the instrument reliability, we do not have a comparison, but this also depends on the maintenance work you do. Any nice instrument will break down if the maintenance is bad or not existent at all...
You could also check supercritical LC. Rather expensive, but the solvent is CO2 which simply evaporates and you do not need to freeze-dry your samples. Also available in preparative scale.
Good luck, Marcus
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I analyse SSRIs (pKa values of them between 9-10) in human plasma by HPLC following liquide-liquide phase extraction. The residue was resuspended with mobile phase (0.25 ml), water: ACN (42:58) (pH:3.5, adjusted by TFA, isocratic), vortex 15 sec., ultrasonic bed 2 min. then vortex 15 sec. The recoveries were lower than 40%. Same tubes were resuspended with 0.25 ml DMSO and re-analysed again and the additional recoveries were more than %40. So, the mobile phase does not solve all residue of analytes from the bottom of the glass tube. I think mobile phase is not always an ideal solvent for resuspension!
Is there any relation between pKa values of molecules analysed and the pH of solvent used for resuspension of residue after extraction?
Thank you
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In agricultural chemistry, we often face much more difficult matrices than plasma samples.  The matrix we encounter frequently prevents complete dissolution of the residues of interest.  To circumvent this issue, we typically dissolve samples with vortex mixing in a small amount of organic solvent (50-100 µL of MeOH or ACN) followed by aqueous mobile phase to a final volume of 1 to 2 mL.  This usually allows for analysis of compounds with extremely different polarities in the presence of extracted plant and animal tissues.  By keeping the percent organic at or below 10%, there is usually minimal peak shifting (any peak shifting can be readily accounted for by dissolving standards in the same solvent).  We frequently use this technique to dissolve samples even when the analyses require large injection volumes (900 µL).       
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I am running reversed phase preparative TLC. After I run the plate and scrap the silica with the desired band, I suspend the silica in methanol and filter it off. The NMR shows impurities around (0.7-1.3 ppm) that did not show up in the crude material. Is this coming from the C-18 modified silica? If so how can I avoid that ?
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By NMR it seems that there is unbound C18 material (or mineral oil) on the plate. I have seen this especially on older plates; it is either unbound C18 or oils absorbed onto the plate (vacuum pump oil from processing the plate during manufacturing?). Mark the top of the plate and elute the preparative reversed phase plate with chloroform or methylene chloride all the way to the top end of the plate. This will concentrate hydrophobic things like C18 residues at the top of the plate. Dry the plate at room temperature, do not heat or put under vacuum!  Then run your sample in the same direction on the plate. This should give you a cleaner background for the NMR spectrum of your eluted band.
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I was looking for a  method to detect and measure oxytetracycline by rpHPLC. I have read many methods that use C8 or C18 columns in combination with  formic, oxalic or TFA acids and acetonitrile as a mobile phase.  I would like to know what methods are generally considered to have the most  successful results ?
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Hi, I think there are many publications for analysis of oxytetracycline by HPLC, see Tabel 3 of our chapter; unfortunately I do not have soft copy of the published version, I just have the proofs, but I t think you can still read it.
Best regards
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I need to quantify folic acid in serum samples using LCMS. for that i need to get a lowest LOD ~ 0.1ng/mL. 
Now we are in a process of running the standards and we got clear peak up to 10ng/mL. Though we got the clear peak the response is varying (3000-6000) within first and next injection even not removing the vial from the tray (Poor repeatability) and also we are getting nearly a response of ~2600 for the blank (methanol) . 
the mobile phase is 30% acetonitrile and 70% water. fflow rate 0.5mL/min
the column we used is XTERRA,RP8,5um,3.0x150mm,
the folic acid is dissolved in water and final dilutions are made by methanol as per the protocol.
Can any one help me to improve the sensitivity of the LCMS for folic acid?
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No need to concern yourself with "improving" sensitivity at this stage if so far you are having reliability problems. First you must obtain reliable and reproducible results. Since your initial observations saw large variations between two injections from the same vial, you will need to start with the basics first. For example: Are you injecting a volume that is within the range of the injector? What is the injection volume? Is it too small or too large? Is liquid evaporating from the vial? Is the cap on too tight (causing vacuum to form, resulting in less sample being drawn the next time)? Have you tested the system with stds (forget the MS, just use a UV/VIS detector) to verify that you can obtain excellent results and reproducibility before you start looking at the dozens of settings on the MS side. Troubleshooting is a logical, step-wise process. First verify the hardware, column, method and techniques used (i.e. how you prepare samples), then check one thing at a time to find out how to obtain a good quality method. Once you have that, then look at the MS portion.
Regarding sensitivity: With a high quality LC-MS method, next optimize the method used and all of the MS settings to obtain low noise and high signal. Heat, gas flow, capEx voltages and so on. Ask yourself if your LC method is hurting or improving the sensitivity? Don't use chemicals that suppress ionization (e.g. TEA, TFA). Don't use non-volatile additives. Formic acid is a great acid to use in Pos mode. It replaces acetic acid and improves ionization. How about using an adduct to improve the response?
Most importantly of all, get help from your own university's LC/MS expert to assist you in developing a method and running the samples. Learning LC/MS takes several years and I suspect you do not have that extra time available, so enlist some help to get the job done. This is to be expected as learning to run LC-MS methods is not like learning to use an analytical balance. I am sure there are people there to help you.
Additionally, I have attached a web link to a page with some free articles that may assist you. I teach HPLC and LC/MS method development and optimization professionally for a living. The link has many articles and topics that I present in my classes. Some may apply to your work..
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I`ve read some papers, and I got one method below:
1. Sample is centrifuged at 2500 rpm in 10 minutes
2. The clear supernatant is collected and acidified 1:1 with 4% H3PO4
3. One μL of acidified supernatant is injected into the liner in front of column
Can you give me some advices?
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VFAs are hard by HPLC. Not a good choice. GC-FID works quite well. Use a wax column or other very polar phase and you won't have to derivatize.
We've run them by both direct injection and headspace. I prefer the headspace method. You would put a sample into a headspace vial (say 5 mL for a 20 mL HS vial) and add 1 mL of an acidified saturated salt solution. You need to get the pH of the sample down below 3 to get them all into the headspace efficiently. Heat the vial to 80C, allow it to equilibrate (say 30 minutes) and then extract a volume of the HS and inject it. We typically inject 1 mL of HS using a 20:1 spilt ratio with a Restek RTX-Wax 20 meter column. You'll get better results with the capillary (you can adapt your packed inlet) but it will certainly work with your packed column and you can run under very low split conditions. 
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Usually and in most cases peak area is used for analysis in HPLC, but in some methods (and also in few ones in USP) peak height is used. I am wondering if peak height can be used for HPLC analysis.
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It can be, but is not the "norm". For HPLC, area is the preferred technique as it does a better job with peak shapes that are not perfectly symmetrical. Peak height can be very accurate if baseline noise is low and you have a gaussian shaped peak (close to perfect, which is often the case in well developed GC methods).
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Polyoxometalate- XRD -Keggin structure
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Your best bet is to look through a crystal structure database and generate calculated powder patterns. The peaks in a PXRD spectrum will vary depending on how the Keggin units pack.
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Having synthesized PHA , I see that the retention time of PHA during a HPLC run cannot be compared to that of PHBV standard as due to obvious diifference in the molecular weight and hydrophobicity the retention varies. 
I also converted the bioplastic to crotonic acid and had run the samples, but I see that conversion and elution of crotonic acid sample of both PHA and PHBV had not much of difference. ( as both are converted to crotonic acid , which doesnt impart a difference in the elution)
What can be fixed as a standard when fermentatively synthesized PHA is to be run in a reverse phase column ? Is it necessary to fix a standard if the sample is to be run in a reverse phase column apart from using an organic acid column ( after being converted to crotonic acid)?
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Other authors that tried to measure polyhydroxyalkanoate composition by HPLC technique used an ion exchange column. Maybe your problem is you are not using the correct column.
In the following lines you can find the method used by Karr et al. (1983) to determine PHA using a HPLC:
Digestion of PHB and Aminex-HPLC analysis of crotonic acid: Samples ranging from 0.01 to 500 mg of PHB-containing material were digested in 1 ml of concentrated sulfuric acid at 90°C for 30 min. The tubes were cooled on ice, after which, a 4-ml volume of 0.014 N H2SO4 was added with rapid mixing. Before analysis by HPLC, samples were diluted an additional 5- to 100-fold with 0.014 N H2SO4 containing 0.8 mg of adipic acid per ml as an internal standard and filtered through a 0.45-,um HAWP membrane filter (Millipore Corp., Bedford, Mass.) to remove particulate material. The injection volumes ranged from 10 to 50 p.1 or sample concentrations from 0.2 to 560 ,ug/ml. Samples were eluted with 0.014 N H2SO4 at a flow rate of 0.7 ml/min from an Aminex HPX-87H ionexclusion organic acid analysis column (300 by 7.8 mm) (Bio-Rad Laboratories, Richmond, Calif.) preceded by an ion-exclusion guard column of Aminex HPX-85X. HPLC was performed with either a Waters Associates 6000 A solvent delivery system with U6K injector or a series 3 chromatograph (The PerkinElmer Corp., Norwalk, Conn.) with a variable loop injector (Rheodyne, Inc., Berkeley, Calif.). Absorbance of crotonic acid was measured at 214 nm (Waters 441 absorbance detector) or 210 nm (PerkinElmer LC-55 B detector). The amount of crotonic acid produced from PHB was calculated from the regression equation derived from known crotonic acid standards.
I hope this answer help you to solve your problem
Best regards.
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i need to know the particular method by which i can deposit droplets of particular chemical reagents on chromatographic paper manually. Can we use office inkjet printe for this purpose? i want to deposit 1% iodine solution (droplets ) on chromatographic paper.
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Dear Research Scientists
I process of algae, I am positive I have produced Vitamin B12; HPLC analysis shows we have significant amount of our product. The problem is the product is not 100% pure since we have carbohydrates, lipids and fatty acids even proteins all are exist in our sample. In process of purification we know by role of thumb, we need column chromatography for separation of Vitamin B12; while having adsorbent such as Ambelite, Sephadex gel, Agarose, Alginate or even molecular sieves to remove impurities. Our objective is to deliver pure Vitamin B12. Separation and purification must be carried out by suitable solvent. In final stage we should remove solvent and deliver 99% pure product for human use. This means we should use green chemistry solvent; avoid any toxic solvent in extraction process.
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Thanks Joseph, our green solvent for super-critical extraction is water. Still we need further purification for fine product delivery.
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I prepared the standard by aliquoting 100 uL of ten different lipid standards (PA, PG, PC, PE, and PI). The lipids were dissolved in solvent either in methanol or chloroform. After aliquoting 100uL of each standard, I dried the mixture in nitrogen stream. The dried lipid were completely dissolved in 3mL of chloroform and stored at -80C. When I took them out after several hours, the standard mixture was frozen. I thought the chloroform dissolved lipid should not have frozen like in water. Is it normal or something went wrong? I dried the standard completely and dissolved in chloroform.
Thank you for your answer.
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Thank you. Sorry, I should have checked that. I blindly assumed that organic solvent should have melting point below -100C or something which is not true. Thanks again.
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Right now I'm doing assay analysis methformin and glimepiride combination using HPLC. I'm using C18 and C8 column.
I have trouble that metformin always in dead time because metformin is very polar,I already use ion pair and metformin still in dead time. 
I'm open with any advice. Need your help
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ANS:- Dear sir,
This method may help you,
The development and validation was carried out by using HPLC (waters) separation 2996 series, variable wavelength photodiode array (PDA) detector module equipped with auto-sampler with injection volume 20 μl, 2693 pump, column used was Inertsil ODS 3V (150 × 4.6 mm, i.d., 5 μm) column,
mobile phase consisting of 0.02 M Phosphate buffer adjusted to pH 2.5 using dilute orthophosphoric acid (solvent A) and acetonitrile (solvent B) was set with gradient programming for 18 min was optimized at a fl ow rate of 1 ml/min, 230 nm wavelength, injection volume of 20 μL and ambient temperature was maintained during the entire process to obtain symmetric peaks of MET and GLI.
2.72 g of potassium dihydrogen orthophosphate (0.02M) dissolved in 1000 ml water, adjusted to pH 2.5 using dilute orthophosphoric acid and fi ltered through 0.45 μm membrane fi lter. Mobile phase B: Acetonitrile Diluent: Water: acetonitrile (50:50).
Table 1 Time programming of gradient elution Time Mobile phase-A (0.02 M potassium dihydrogen orthophosphate buffer) Mobile phase-B (acetonitrile) 0 -80: 20 3- 50 :50 5 -30 :70 12- 30: 70 15- 80: 20 18- 80 :20
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I want to quantitate and annotate the features obtained via QTRAP5500. Can I use it for the data acquired through direct injection or I need to get data acquired through LC-MS?
Thank you.
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I believe you can use multiquant for MS data acquired by flow injection analysis. The problem is that your are using a low resolution triple-quad, many species within the 0.2 amu mass resolution of the instrument can co-elute in the injection slug and you will not be able to tell them apart using the 5500. Using chromatography up front gives you the potential to time resolve many of these compounds that are close in mass and detect  (identify) them separately.
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I'm using a C-18 column to detect the acid (neutral and succinate species), but when I use a binary mixture with glucose the sugar elutes almost at the same time with the succinate specie. It also seems that this column is not the best one for glucose detection, 'cause I'm not able to make a good calibration curve. 
I'm using UV detector at 210 nm wavelengh. 
I'd like to know what kind of column can I use for both compounds or other way to separate them.
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Dear madam,
This chomatograpic conditions will help you,
AMINEX HPX-87C(250x4mm) column is used  for carbohydrates, monosaccharide’s,beverages analysis & amino column with  ELSD detector or RI detector .
eluent:-30% ACN
column Temp:70 0C
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Dear Experts,
Attached files are the mass spectra for blank run (methanol:chloroform 1:1). I got so many noises. Is that contamination or spectra for mobile phase (Acetonitrile, Methanol, etc). I tried to flush using different gradient concentration of mobile phase but the LC-MS was not clean. 
Could you please explain me about the problem observed in mass spectrum file? How can I get rid of contamination from the lipidic compounds?
Thank you.
Sangeeta
Begineer for LC-MS
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Bill is right, more information are needed.
Additionally you may check if your solvents are contaminated (use a fresh bottle), clean everything well prior change anything. Also clean the spray chamber/check for dirt. You may also clean the MS capillary which connects the spray chamber with the internal part of the MS instrument. It yould be a contaminated needle, tubing, whatever had contact to something which hardly dissolves in your solvent system and flushed out only slowly. But this is just guessing!
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How to elute protein bound strongly to the beads? Does using loose beads helps or I should optimize the buffer solutions etc?
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Can you be more specific about what kind of column you are using? Is your protein soluble, does it have an extreme PI, form quaternary stucture, or some other factor that could lead to unusually high or low binding affinity for the column?
Firstly, it is always a good idea to check and make sure your samples actually contain your recombinant protein in concentrations that you expect. I use IPTG-inducable expression and run an SDS-PAGE gel containing uninduced cells, induced cells, lysate, and a sample from each wash as well as the elutions. You can Western blot this gel to ensure that you are in fact obtaining protein, as poor expression may be the culprit for lower than expected purification yields.
You should also ensure your protein is soluble in the buffer you are using. I use bioinformatics calculations on my raw sequence files to estimate the pI and stability of my protein and design my buffer around that. Tris, sodium phosphate, HEPES, and sodium acetate are some common buffers that operate in physiologically relevant pH ranges. To stabilize proteins, sodium chloride is also often employed in the range of 50-200 mM. Generally speaking, more salt is better for stability but this is a case-by-case basis as every protein is unique. I use a Dynamic Light Scattering (DLS) instrument when I am buffer-optimizing to check for polydispersity and aggregation that can be indicative of poor buffer compatibility. Filtering solutions and/or spinning them and discarding any insolubles helps prevent additional protein loss to aggregation.
If these don't help, IMAC columns are eluted using concentrated imidazole washes (250-500mM). If washes are insufficient, I commonly incubate the column resin in my elution buffer and suspend the beads to maximize competition for the column resin. Alternatively, If you are using the column on LC instrument, try reducing the flow rate. As mentioned above, ion exchange columns are eluted with high concentrations of NaCl and the same principles would help improve your elution efficacy if you are getting low yields from the column.
Lastly, if your protein is for some reason still present in high concentrations on the column, it may require stripping of the column resin to remove. You should refer to the product manual for the specific protocol that is appropriate for your column. This may occur if you attempt to purify multiple His-tagged proteins on a single IMAC column, and so this is not advisable. Note that protein removed from a column in this manner is not usable, but regeneration of the column resin may help with purification yields. Most manufacturers recommend that the column be cleaned every 2 to 10 uses, depending on the nature of its use.
I hope this helps, and good luck!
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I'm try to gradient HPLC with Water/ACN including ammonium acetate, acetic acid(after i'll call ammonium acetate, acetic acid as two chemicals.) Using reverse phase C18 analytical column from Hamilton
my experimental procedure : water 80/ ACN 20  is starting condition -> sample injection -> ~5 min water 80/ ACN 20 -> gradient to water 20 / ACN 80 until ~25 min -> ~40 min water 20/ ACN 80 -> ~50 min returning to starting condition. (these two buffers mixed by HPLC pump)
Detection wavelength is 220 nm. 
I attached my HPLC results by image file ( with Five different condtions)
When first i used only pure water & ACN. There was NO Baseline drift.(blue line data.TEST 20)
But when i added two chemicals into water solution. then there appeared baseline drift!!(TEST 21~23)
Test 21 (water with 5mM ammonium acetate + 0.05% acetic acid)
Test 22 (water with 5mM ammonium aceate)
Test 23 (water with 0.05% acetic acid) 
baseline drift looked like synergic effects by two chemicals(when they added alone, they caused baseline drift. but added together, there was much bigger drift.)
I tested other condition. I made two buffers with mixed form (SolA : Water+ 80 / ACN 20 & Sol B : Water+ 20 / ACN 80, Water+ : Water with 5mM ammonium acetate & 0.05% acetic acid) SolA is starting buffer(100%) and SolB is increased conc buffer by gradient.
But there also had baseline dirft (TEST 25)
I have NO other idea for removing baseline drift :(
I'm using all kinds of buffer or chemical with HPLC or LC grade from JT BAKER or SIGMA ....
Please help me
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Here are two good recommendations for novice to intermediate level users.
"Introduction to Modern Liquid Chromatography"; by Snyder, Lloyd R./ Kirkland, Joseph J./ Dolan, John W.   ISBN 0470167548
"Practical HPLC Methodology and Applications"; by Brian A. Bidlingmeyer. ISBN 9780471572466
  • Additionally, here is a link to a website with lots of free HPLC "Hints & Tips" to help you learn about and become a better chromatographer.  http://hplctips.blogspot.com/
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What would be the best alternative for automated alignment of GC data of complex mixtures?
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It depends on what kind of data you are handling... 
BTW you might have a look to MetaMS.
Enjoy
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I am trying to develop an isocratic method for separation of a number of impurities in an antibiotic sample. Its just an idea if someone can guide me whether I can use both the ACN and MeOH collectively in an isocratic mobile phase....?
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Methanol is a very common choice for the HPLC mobile phase organic solvent component in reversed phase HPLC even though acetonitrile is often superior in several aspects. On the other hand, methanol is often less expensive and less toxic than acetonitrile, so it has advantages as well.
If you wish to simply substitute methanol in place of acetonitrile in a Reversed Phase HPLC method that has already been developed with acetonitrile, you should be aware that methanol is a much weaker solvent than acetonitrile and therefore you will not get the same chromatographic results. For example; if you have a method with 20% acetonitrile and then change it to 20% methanol, you will get longer retention than with the acetonitrile.    
Methanol is more polar than acetonitrile so it will increase retention of hydrophobic compounds in reversed phase and the elution order for some analytes could change as well, especially when a Phenyl Hydride column is used. For these reasons, changing to methanol should be viewed as a method development tool and not as interchangeable with acetonitrile. Also, for many SOP's changing from acetonitrile to methanol requires internal approval.
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I am trying to separate some bee venoms by HPLC.  After runs, there is residual protein on the column, even after multiple washes from 5 to 100% ACN.  Although there is no mention of this in recent (last 20 years) papers, older papers say that melittin (a major protein of many bee venoms) aggregates and sticks to columns.  However, there is no mention of how to get it out.  In fact, recent papers don't even discuss the issue at all, as if they are not having the problem.  Does anyone have experience with this?  Thanks!
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As Bruce suggested, you may add some acid to your mobile phase. 0.1 % TFA or HCOOH is commonly used. Be careful with basic conditions, this may destroy your column material. Some organic buffers may help (e.g. HEPES) adjusted to pH 7-8, this increases the solubility of the protein but does not destroy the stationary phase. At first, wash with 10 column volumes 100% H2O, then add 5-10 column volumes buffer at low flow rate (0.05-0.1 ml/min), remove buffer with H2O + 0.1 % acid (20 column volumes), then try the organic phase again (with acid added).
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I am working on addition reaction which generates a two adjacent stereocenters. If this reaction is forming a pair of diastereomers then TLC should show two different spots. But I am getting a single spot on TLC. Does the diastereomers always show a different spot on TLC or only in few cases? Please explain.
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Whether you get a single TLC spot for a mixture of two diastereomers or not depends on how differently the diastereomers interact with the stationary phase and the mobile phase. If you get two spots, you have two diastereomers. If you get one, you have to keep trying. One spot does not mean you have only one diastereomer. Predictions are difficult.
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I am wondering how to calculate an optimum flow rate depending on the dimensions of the column in RP-HPLC?
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Are you looking for the complex calculation (using the van Deemter plot curve, which can be obtained for free online or from one of the wonderful books on chromatography) or just the main guidelines we use in practice to develop methods? A formal plot and calculation can be made which takes into account the particle size and efficiency to determine the optimum flow range. But there is a better way for everyday purposes.
For practical considerations, to maintain linear velocity we use 1.00 ml/min for 4.6 mm ID columns, 200 ul/min for 2.1 mm ID columns and 50 ul/min for 1 mm ID columns.
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I'm purifying a protein and to prevent it from aggregation, we should use high concentration of 2-ME (700mM or 4% w/w).
but when we use this concentration of 2-ME we have serious problems in equilibration, sample application and elution.
for example in equilibration we should use over 15 CV buffer to fully equilibrate the column while this becomes to 4 CV when we don't add 2-ME in our buffers.
or in sample application, our protein attach less than when we don't add 2-ME.
and our peaks' shape in elution is abnormal in comparison to when we don't use 2-ME(they have long tail).
now our question is what's the problem?
Is there any interaction between 2-ME and columns?
our columns are: DMAE Fractogel and TMAE Hicap Fractogel.
buffers are : 20mM Tris + 4% 2-ME w/w and 20mM Tris + 500mM NaCl + 4% 2-ME w/w
We will appreciate for your help.
Thanks.
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Have you tried other reducing agents besides 2-mercaptoethanol to keep your protein from aggregating? (Is it highly prone to intramolecular disulfide bond formation?) It is more usual to employ 1 mM ditiothreitol or TCEP to keep the protein reduced.
Here is a reference that deals with prevention of protein aggregation during purification:
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when I think of size exclusion chromatography for peptdies/proteins, I consider aqueous mobile phase containing buffer ions, salt, L-arginine etc. But, in the USP monograph for Insulin (http://www.pharmacopeia.cn/v29240/usp29nf24s0_m40520.html), the innovator has used acetonitrile and acetic acid for HMW estimation.
Can anyone tell me the possible reason for using such solvent system and its effect on chromatogram if I change the proportion of components? 
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Now I see what the USP is doing.  The "L" is their designation for HPLC columns and this is actually a LiChrosphere resin which is a good SEC media for the separation of smaller protein and peptides by that modality;  better than the soft gel media which have some difficulty in this smaller molecular weight range and utilize highly aqueous solvents.  In this case, to take advantage of the SEC functionality but avoid any other interactions of the resin with protein, the method in "Limit of High Molecular Weight  Proteins" calls for a solvent of acidified aqueous arginine with a 20% acetonitrile  organic modifier to prevent insulin and its oligomers from binding to the resin while the acetic acid rather than TFA acts as the ion pairing agent and acidifier for the usual silica-based media stabilization as well as insulin solubility/stability.  A diol bonded phase is very much less hydrophobic but that doesn't mean the absence of any hydrophobic character due to the bonding chemistry that attaches the diols (Journal of Chromatography A
Volume 915, Issues 1–2, 27 April 2001, Pages 35–42 ).  The arginine acts to prevent any further oligomerization through the effect of the guanido group.  TFA is a stronger acid than acetic acid and more denaturing and is not needed since this is not reverse phase chromatography.  
Indeed, reverse phase resins and solvents have protein denaturing effects but the smaller molecular weight proteins that have multiple disulfide bridges, high ratio of bridges to mass, maintain tighter conformations under these conditions compared to other proteins with relatively larger mass and few disulfides.  Not only insulin but growth factors (protein hormones), cytokines (chemokines, lymphokines), TNF family members, TGF beta family members, etc are examples of relatively small molecular weight disulfide-bridged proteins that have been purified by traditional preparative reverse phase chromatography and, after removing the acetonitrile and TFA as though the proteins were small organic molecules, the proteins are fully active.  You might be interested in the CD data of these proteins in these aqueous acetonitrile solvents.
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Does anyone here have an established guideline on conducting HPLC on plasma sample? How much of standard solutions (containing serial concentration of drug) to be added into 500ul plasma prior analysis to obtain the standard curve?
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thanks so much for answering my question. Is there any reference based on your suggestions?
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I have a non polar column DB5ms ul GC column. But i am unable to find a protocol for  standardization and estimation of VFA using the column.
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Jaya, probably you wont find a GC method using a non polar column for those analytes since the phase of this column has low polarity and works fine with another kind of semi-volatile samples. I  would recommend you use a polar column, the best for VFAs is HP-FFAP, it works pretty good (C2-C6) , great resolution and sensibity, however you can use a regular polyethylene glycol phase column. 
Other solution could be the alcoholic derivatization of VFAs, not  highly recomnended because the losses in the procces but you can try if you can't chose your column.
Regards.
Alexandra 
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I developed a RP-HPLC method to separate five compounds (diphenyl iodonium triflate, triphnyl phosphine, triphenyl phosphine oxide, tetraphenylphosphonium tirflate, and iodobenzene) at concentration of 1mg/mL in acetonitrile using a Waters C18 column (3.5µ with 2.1×30mm). The mobile phase graduates from 100% buffer of 20mM ammonium formate to 100% acetonitrile within 5 minutes and I have got a very nice separation (the chromatogram is appended).
Then I decided to extend my chromatography experiments and I bought new column (similar to the previously mentioned column).
Unfortunately, the new column did not behave similar to the old one in which its response much lower than the old one (for example the response drops by 100 times for the same compound at same concentration , i.e. if the response of the old column is 250 mAU, it becomes about 25 mAU for the new column). Eventually, the pressure significantly increased, and then the column got blocked.
I tried to solve the problem according to a procedure stated on Agilent’s website (https://www.chem.agilent.com/cag/cabu/ccleaning.htm), but it has not succeeded in solving the issue.
ِAnyone can advise me to solve this problem?
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1st. Apologies for an earlier typo. Your column void volume is ~ 80 ul (not 8ul as it initially appeared from the system truncating the character). At 1ml/min your Tzero would be ~ 0.08 min (4.8 seconds)!
Saddam: I feel that you would benefit the most if you could find someone at your University with proper chromatography training who could help you in this matter. You appear to be going about this method development process without any understanding of chromatography fundamentals and such an approach will result in poor quality data. You still seem focused on column cleaning and receipt of defective columns. I think your efforts would be better spent understanding the technique used first. Once you have gained an understanding of the technique and used the skills to develop a better method, then you can evaluate columns. As someone new to the field you can not expect to master this technique, the use of the instrumentation or even the software without first having the benefit of months and years of proper training first. - And then, years of practical experience with samples. Many scientists using this technique still do not have a good understanding of the technique and how the many parameters affect the data, even after many years of use. It is a complex analytical technique that takes time to master. Having an experienced professional on hand can greatly help guide you through this process as can reading many of the wonderful books on HPLC principles too. "We" can only provide so much guidance to you via this forum. You need more help with the basics and fundamentals first for our comments to be of value. I hope you have someone there who can provide training to you.
There are too many individual problem items in your method and description which need optimization to list here.  However, I wish to comment on one basic area that I hope you will think about and adjust when you are re-developing this method for the samples.
Solubility: You wrote that all of your compounds do dissolve in pure ACN, but none of them dissolve in your buffer solution. Yet, you start your gradient method with 100% buffer!  The same liquid that none of your samples dissolve in. You also mentioned that when you started the gradient at 95% buffer, you had very poor results so changed it to 100% buffer. This makes no sense at all.
I will leave you with a link to a free webpage which contains many short articles on a variety of chromatography topics. All are focused on teaching and stress the importance of learning to use good chromatography fundamentals. I hope you find it valuable as you begin to learn this wonderful analytical technique.
Good luck to you.
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This is long, sorry. I plan to use LC-MS to determine the concentration of several small molecules. In set of experiments these will be (i) derivatised sugar phosphates (using AEC) and in the other case they would be (ii) really small peptides (di, tri, tetra...).
Initially I think using an external standard would be the best option as I was planning to use calibration curves for each analyte and just use a pot with only the external standard on it every time I do a run. As far as I know this would be a valid thing to do and the use of an external standard would minimise the random variations of the LC-MS machine. Questions:
(1.1) If an external standard would be good, should I use the same one for both types of analytes (sugars and peptides)?
(1.2) I reckon the standard should be stored in solution so it should be very stable, or maybe it can be aliquoted and frozen so every time I would only thaw one of them?
(1.3) Which actual substance would you recommend?
Despite this, my MS technician insists that maybe using an internal standard would be a better option as, according to her, if I use one I could use it not only to minimise the variation caused my the machine but also I could use its signal to directly quantify my analytes (without the need of calibration curves). My concerns about this are:
(2.1) I guess I would need to find two different internal standards, one for each type of my analytes as their ionisation/fragmentation properties would be completely different and the internal standard has to be very similar to the analyte you want to quantify, right?
(2.2.) If you think an internal standard would be the best option, any ideas of internal standards for (i) sugar phosphates and (ii) small peptides?
Thank you all!
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The choice of an internal standards very much depends on the actual problem you are trying to solve (which analytes in which matrix ...).
Generally speaking isotopically labelled standards are the best choice if you want to account for matrix effects hampering your quantification. Often such labelled substances are not availavble, so one would have to think about a substance which has a chemical structure very close to the actual analyte, but at the same time will not likely be present in the sample matrix.
In case the matrix of your samples does not show a high variability, it might be good enough to use an external (but matrix matched) calibration with a recovery test now and then in your sample series. 
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Hi everyone,  we are having an issue with our Dionex ICS 5000.  We are analysis phenol levels in alcohol samples from different distilleries within the company using HILIC and electrochemical detection (glassy carbon electrode) and gradient elution.  We were running our samples very happily on an old Dionex ICS 500 machine, but about a year ago we upgraded to the ICS 5000 and have had issues with peak height in our phenol analysis ever since.  Basically our peak heights sequentially drop with each sample we run until no peaks are detected (this only happens with phenols and not with our carbohydrate analysis on the same system but with a different column and eluents).  We have tried everything from multiple different cleaning methods for the glassy carbon electrode, changing both the reference and glassy electrodes, changing the detector, buying new reagents, changing the column, and getting thermo involved and nothing has worked.  We are now back to using the old ICS 500 as it has never had this issue.  Thermo don't seem to have any idea of what could be the cause either.  Can anyone help?  Many thanks
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Hi Angela,
I am so pleased that you resolved the situation.
Kind regards,
Ade
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Neem powder is packed in column and water containing Fe (III) is continuously running through column. After process of cleaning water, we want to reuse this neem powder. We want to now what is best eluting liquid for this neem.
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I wonder wether or not it is clever to use water to wash of Fe(III) cause it may cause precipitation of iron-oxyhydroxides that form as a consequence of Fe(III) initiated hydrolysis. Is one of you aware of negative effects of this precipate formation? I imagin some sort of coating may form, that prevents an effective desorption.
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I am doing a research about the effect of beta-glucosidase from almond on the hydrolysis of quercetin-3,4'-O-diglucoside and quercetin-4'-O-glucoside in onion.
But i'm encountering some difficulties in the methodology. for now, i refer to the methods from some of my senior(who graduated long time ago). I wanna do a deeper research about the methods but i hardly  find any published papers about it.
here is the method i used:
10mg of the powder form of the beta-glucosidase is added (10U/mg) to 150uL of PBS(pH7.0). then added 50mg powdered onion extract(extracted by acetonitrile) which is dissolved in 150uL of the same PBS buffer. then let it react for 3 hours in a water bath of 37C.
After 3hours, 300uL of 10%TFA is added to cease the reaction and the whole mixture is centrifuged. supernatant (around 300uL) is analyzed by HPLC and compare with the HPLC result of the control without enzyme.
however, it seems that PBS and TFA doesnt dissolve the glucosides so well...so the HPLC profile doesnt show the peaks that i used to see when i used DMSO to dissolve the onion extract. but I feel hesitate to add DMSO to the emzyme mixture because I suppose DMSO will suppress the enzyme activity.
what solvent should i use to do this experiment so that it doesnt interfere with the enzyme activity while flavonols content can be analyzed with HPLC  ?
or is it possible to use other method to compare the flavonol content without HPLC?
I am a beginner in research. any suggestions and opinion is very much appreciate. thank you everyone.
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I think you can slightly increase the pH of the enzyme reaction mixture to enhance the solubility of quercetin glucosides if your enzyme can keep its activity in higher pH (pH 7.5 or 8.0). Besides, you can try to use glycerol solution (25% or 50%) to replace DMSO if your enzyme is not sensitive to glycerol.
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I need to buy a LC/MS system (single quadrupole).
Two suppliers offered us such a system: Waters (ACQUITY UHPLC SQD); Waters Arc/Qda System and Agilent Hplc 1100 with 6120 SQ;
In your experience, which company's product is the most robust, sensitive and reliable?
Thanks in advanges fopr your help
Francesca
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Hello Francesca: A few general comments, but in general we can not (and should not) tell you which system would be better for your own applications. Everyone's needs are different and their is no one "perfect" system out there. Other individual's experience may or may not be relevant to your application. Both Agilent and Waters offer good HPLC and single Quad systems. Either of them would be fine. The real (and perhaps more important) difference will be found in the experience of the operator and the methods used, not the instrument. Training in how to properly maintain and run both the HPLC and MS system is far more important, than which system is selected. IMHO: All operator's should know how to perform PM's and logically troubleshoot the flow path and system for problems (critical to daily operation).
One item that I would personally insist on is that the HPLC be equipped with a diode array detector (DAD/PDA). LC/DAD/MS is the most common configuration found and allows for the maximum amount of useful data. Each detector's very different, but complementary detection system allows for a wider range of sample types and they are also very helpful for troubleshooting problems found (both instrument and sample).
Perhaps it is possible for you to demo the systems? If so, you could use the time to "see" and get some hands-on time with the software and hardware Maybe run a few samples too?
If you have not done so already, please obtain professional training in how to best use the instrument you select, after installation. If possible, have the training on-site, for several days of hands-on experience. You should learn on your own system, not someone elses (as it will be different). Make sure that the people responsible for running the system on a day-to-day basis are in attendance. Both the software and hardware need to be considered during training, as the learning curve can be steep if you do not have several years of experience using these types of systems.  *Each LC/MS instrument has specific functions and steps that must be learned properly to operate it well. You will learn these and other important tasks much faster having the training done in this fashion.
Also, if all of the service and support for the instrument are to be provided by the same vendor, then be sure to inquire with others working in the same area as you about what their service experiences have been with the vendor. Most vendors provide great service in some areas, and not so great service in others. This question should be asked well in advance of any purchase.
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I and doing some parallel reaction monitoring experiments. I have a large peptide (25 aa). I am looking for the endogenous form of it in whole cell digest and I have spiked in and synthetic heavy labelled form of the peptide too.
Generally speaking the retention times of both should overlap. However the spiked peptide eluted at  48.8 min on my gradient. The best peak I am getting when looking for the unlabelled endogenous form is at 38.8 min on the same gradient.
Is that possible or even believable? I have never worked with such a large peptide before, I can't imagine that the fragmentation would be all that good.
Any help or insight would be appreciated.
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Dear Juhura
You do not really give much information and there are many parameters that can affect what you are describing.
Taking the difference in the retention time between the two peptides (endogenous verses synthetic) I would suggest that they are not the same peptide. The retention times are way different.
How did you come to the "best peak" conclusion? Is this based on peptide mass? What criteria are you using to assess the different peaks?
How are you monitoring the eluted peptides, UV or mass spectrometry? The elution profile of peptides can be very complex and the question is how did you do your sample preparation? How did you get rid of the other compounds in your matrix like nucleic acids, lipids, carbohydrates, proteins, salts etc
Did you run the synthetic peptide as a standard in a separate injection or was it spiked into the cell lysate and if so at which point of the sample preparation?
How are you processing and enriching your sample? Is the cell lysis by detergent or mechanical means? Do you add protease inhibitors for the different classes of proteases?
Is the peptide prone to disulphide bridge formation, in which case there could be intra peptide bridges forming, and these could change the retention time of the peptide but this can be prevented by acetamidation.
If using mass spectrometry to monitor the eluent, then the mass should differ by the number of heavy isotope atoms added during the synthesis of the peptide as the precursor mass. If running MS/MS the fragmentation should be the same with the same mass offset seen for the precursor up to the labelled amino acid(s) after which the masses should exactly coincide.
If the peptide from the whole cell digest is eluting early it would imply a more polar peptide, which could point to a truncated peptide due to cleavage by a proteolytic enzyme during the lysis and sample preparation. This is an often overlooked aspect of sample preparation and when using cells or tissue can be a major complication.
If you have enough heavy peptide you could try adding it to your sample before your cell lysis and check if it also shifts in retention time, but this is a rather expensive experiment, taking the cost of isotope labelled peptides into consideration.
Fragmentation of longer peptides can be quite good depending on the amino acid sequence and ionisation state during mass spectrometry. 
More information could help trouble shoot your problem.
Good luck.
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RBC fatty acid via GC-MS ?
Non-fasting venous blood samples were collected in Vacuette® tubes containing
EDTA. RBC were isolated immediately by centrifuging whole blood at 3000 rpm
for 10 min at 4°C. They were then washed three times with isotonic saline solution.
The RBC samples from Cohorts 1 and 2 were divided into two portions before they
were frozen at -20°C and stored. No antioxidant was added to the RBC samples
from Cohort 1 before they were frozen at -20°C. The antioxidant BHT dissolved in
methanol (500 mg/L) was added to the RBC samples from Cohort 2 at a final
concentration of 10-20 mg/L before they were frozen at -20°C. The duplicates were
lipid-extracted and their FA composition analyzed on the same or adjacent days
within 20 weeks from collection of the blood sample. The RBC samples from
Cohort 3 were divided into seven portions, and BHT was added to defined portions at
a final concentration of 42 mg/L. Baseline portions with or without BHT were lipidextracted
within three hours of collection, and FA composition analyzed. The other
portions were frozen at -20°C and stored until FA analysis after a defined time
interval.
- RBC total lipid extraction
Total RBC lipids were extracted with isopropanol/chloroform (2:1 v/v, Merck,
Darmstadt, Germany) as described by Bligh & Dyer (184), except that isopropanol
was used instead of methanol. The antioxidant BHT was added to the extraction
medium at a final concentration of 50 mg/L. The procedure of lipid extraction and
FA analysis of RBC is shown in Fig. 10. Thawed RBC samples were transferred to
teflon-lined, screw-capped glass test tubes and hemolyzed with distilled water.
Isopropanol was added to the RBC samples (10:1 v/v) and agitated for 45 min at
room temperature. Chloroform was added to the mixture (isopropanol: chloroform
2:1 v/v). Phosphatidylcholine, diheptadecanoyl (PC 17:0) (Sigma Chemical Co., St.
Louis, MO) was used as an internal standard to monitor recovery. After agitating for
45 min at room temperature, the samples were centrifuged at 1700 rpm for 30 min at
20°C. The sediment contained heme and other proteins, and the supernatant
contained total lipids and solvents (monophasic). The supernatant was transferred to
another teflon-lined, screw-capped test tube, chloroform was added (isopropanol:
chloroform 1:1 v/v), and distilled water (isopropanol: chloroform: distilled water
1:1:0.8 v/v/v). After vortexing and centrifuging at 1700 rpm for 15 min at 20°C, the
solution became diphasic with the lower phase containing the lipids. The upper
phase was removed and discarded and the lower phase transferred to a new teflonlined
screw-capped test tube. The extraction medium was evaporated under a stream
of nitrogen at room temperature, leaving the lipids as an oily layer on the test tube
walls.
-  Fatty acid methylation
The RBC total lipids were transmethylated for 45 min at 110°C using 14% boron
trifluoride/methanol (Sigma Chemical Co., St. Louis, MO). Heneicosanoic acid (C
21:0) methyl ester (Sigma Chemical Co., St. Louis, MO) was used as an external
standard. Before methylation, the samples were flushed with nitrogen to replace
oxygen. After transmethylation, the test tubes were chilled at room temperature.
The fatty acid methyl esters (FAME) were extracted three times with hexane. The
combined portions were evaporated under a stream of nitrogen at room temperature,
leaving the FAME as an oily layer on the test tube walls. The lipids were dissolved
in isooctane and transferred to gas chromatography (GC) vials, closed tight and
stored at –20°C until FA analysis.
- Fatty acid analysis
Samples  were analyzed by high resolution capillary GC (HP Series II
5890 A, Hewlett Packard co., Palo Alto, CA, USA) equipped with a flame ionization
detector and a GC-Capillary polyethylene glycol column from Chrompack (CPWAX
52CB 0.32 mm inner diameter (i.d.) x 0.2 μm film thickness x 25 m). The
injector and detector temperatures were maintained at 235°C and 250°C,
respectively. The column temperature was programmed to have an initial
temperature of 90°C for 2 min, then rising by 30°C/min to 165°C and at 3°C/min to
225°C and then held isothermal for 6 min. The carrier gas was hydrogen at 31.8 kPa.
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The method is suitable. The MacLafferty product at m/z 74 - makes spotting FAMEs easy and is always present even if the molecular ion gets lost, which can happen in polyunsaturated FAs.
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Hello.
I want to identify compounds by comparing retention index(RI).
In LRI equation, I have to substitute carbon number and retention time of lower alkane and upper alkane.
So I wonder that if i want C3 compounds, do I use C2 and C4 alkane as lower and upper alkane?
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Thank you Alex, Peter and Santosh!
First, I need to determine retention time of my sample, and compare to alkane.
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The HPLC is plumbed with PEEK tubing and a major component of both A and B mobile phases is chloroform; my analytes are lipid-A type molecules. All reagents and solvents are HPLC/LCMS grade and filtered. I see significant baseline noise and signal variance by charged aerosol detection; what could be the cause?
Thanks!
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PEEK is generally safe with chloroform (per the manufacturer). Should not cause ghost peaks on its own.
These types of detectors are very complicated to use and troubleshoot. Perhaps some of these comments will help you troubleshoot the issue(s)?
CAD and ELSD detectors accumulate sample debris from use. This debris increases the total noise level seen over time. Both types of detectors require periodic cleaning. Be sure to measure the actual noise seen under controlled conditions with a std so you can evaluate any changes over time.
The most common reason for high baseline noise with either an ELSD or CAD detector are through the use of non-optimized detector settings. The settings must be adjusted and optimized for each mobile phase and flow rate. The mobile phase must be 100% volatile. The nebulizer atomization and desolvation temperature must be optimized to the flow rate. This involves adjusting the gas pressure, flow rate and temperature to acheive the best sample S/N ratio or lowest noise S/N ratios. This process can take several hours to perform. Try to optimize the settings, step-by-step, changing one thing at a time to determine the best settings for your application. Failure to 'dial-in' the right settings with these detectors will result in peaks which vary in shape, area and height. IOW: Very poor reproducibility. *A dirty atomizer/nebulizer will also cause similar variability in signal.
Note: When optimizing the detector settings, please do not make changes based only on the observed baseline noise. The noise must always be measured relative to the sample signal.  Higher noise does not always mean lower signal. Lower noise does not always mean better signal. Select a gas flow rate and desolvation temperature (based on the mobile phase's boiling point; see Link provided to table of solvent properties) which removes the mobile phase and results in clean atomization. High noise and/or variable signal will result if the temperature is too high or too low. Same is true with the gas flow rates.
Lastly, make sure the exhaust outlet for your CAD is clean, unobstructed and well vented. Clogs or obstructions here can also induce all kinds of noise and variability.
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Hello, 
Anyone has used a method for measuring sodium chloride using HPLC?
can you please send me the method?
Can that method be qualitative as well? I mean, can I use it to identify NaCl or Cl- in unknown samples?
Thank you very much
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Dear Aurea, 
Thank you for your helpful replies. 
In your opinion, do you consider HPLC to be a qualitative method so we can rely on it for identifying the compounds or it is only quantitative method for measuring the concentration of known compound based on standard solution?  
Thank you, 
Abdel
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I want to quantify 4-NP in HPLC at 245 nm but the peak is observed downwards, under the baseline. How could I overcome this in order to proceed with my method? My mobile phase consists of  orthophosphate buffer and MeOH.
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Dear Efi:
You should see if your standard is stable in the solvent you are using. For studying this, you should prepare a solution of your standard into the methanol/orthophosphate buffer and run the chromatogram each 10 or 20 min for studying their stability. Provided the signal decreases, your standard is not stable into the solvent selected.
Best Regards,
Áurea Andrade Eiroa.
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Hello Friends,
I am working on Lumenfantrine analysis by HPLC as per USP method, but in USP 38 column temperature mentions as Column temperature: Beginning of column, 50°; end of column, 35°.
This created lots of confusion could anyone clarify on this whether this is column oven temperature programming in method or kept different temperature on both end of column which is not feasible.
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Hi Sabine Harrison,
Thanks for your valuable answer.
But is it possible with any system to set different temperatures at column inlet and column out let, if there is any such HPLC then please let us know.
As per my experience with Liquid chromatography it is not possible as column is constructed with metal (SS) and metal is very good conductor of heat. So it doesn't looks feasible to set different temperatures at column inlet and out let. second problem with setting different temperature is length of column as per USP which is 50 mm only.   
In my opinion this should have been performed by programming column oven temperature i.e. by keeping 50°C at start of analysis and 35°C at end of analysis (continuous decrease in temperature like mobile phase gradient) which is a feature of most of HPLC column ovens.
  • waiting for more answers to conclude the problem.
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Hi everybody,
I am using the internal standard technique for building a calibration curve for my samples. I am using different range of concentrations for my samples (15-2000 microM) and the concentration of the IS is 500 microM, constant in all of them. I am using the IS peak area for normalizing the peak area of samples. Everything is perfectly fine, except for the highest concentration, in which the IS peak area decreases (when it should be constant because the used concentration is the same), while the sample peak area does not change compare to the second highest concentration. When I plot the data points, there is a perfect linear correlation except, of course, for the highest one. I have tried to change the range of concentrations used (decreasing them), but the problem is still there, and only for the highest concentration! Have you ever had this kind of problems? I would really appreciate any thoughts/suggestions that can help me... thanks!
Nicoletta
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What you are trying to do is a pretty standard analysis. There is a lot of literature on it using a standard GC-FID with similar columns to yours. About 30 years ago I did some work on long chain alcohols with on older version of your column coupled to a magnetic sector mass spectrometer. After the method was developed on the GC-MS we were able to run it as a routine analysis on a GC-FID. The main difference from your method was that I used trimethylsilyl derivatives, but I am confident that your acetyl derivatization is fine.
OK, some ground rules:-
1) The amount of your internal standard injected on column **must not exceed 100ng**. If you do, the chromatography will be bad - Your peak will front, the geometry and retention time of your internal standard will change (dramatically) with only small variations in solvent composition, polarity and concentration. The 'perfect amount' will be between 10ng and 20ng. Please trust me on this :-) Going over 100ng will not give you any better results, honestly. If it does not work at 10-20ng your method or equipment is badly broken...
2) I am assuming that you will use a splitless injection - If you have a standard split/splitless injector **do not inject more than 1µL of sample** - You can use 2µL if you really, really, have to - But that will only be useful if you are doing trace analysis (you are not).
3) Use an initial column temperature 20C lower than the boiling point of your solvent (heptane b.p. 99C) so your initial temp of 80C should be OK. Ramp the column oven quickly to a temperature of 20C above the solvent boiling point - 30C/min should be fine. Then continue with your normal 10C/min ramp. I would go a higher than 240C (maybe 300C) to make sure than any high boilers elute, otherwise they will stay on the column and come out as random wide peaks in later runs.
4) Set your carrier gas flow velocity to 25cm/sec for helium, or 35cm/sec for hydrogen (1.2 or 1.8mL/min).
4) Do not complicate the method any more than you need to. Avoid Pulsed Electronic Pressure Control. You don't need to use it as you only are injecting 1µL of solvent. The heptane solvent will only produce ~210µL of solvent vapour inside your injection liner (A 2mm ID liner will hold ~250µL, a 4mmID liner will hold >1000µL).
So how do we find out what is going on? If we use simple logical steps it should isolate the problem:-
1) Inject 1µL of pure heptane solvent as a blank. Do you get large peaks coming out in the region of your analyte or IS? If you do they might be retained on the column as dirt or as carry-over from a previous injection.
2) Repeat the solvent blank injection. Have most of the peaks gone? If yes - It is now OK. If not consider cleaning the liner and bake the column off at 300C
3) Now make up a 100µM/L C12Acetate internal standard solution - This is equivalent to 23ng/µL. Run 3 replicates of your 23ng/µL internal. Yes I do mean only 1µL of just the C12 internal standard :-) **Do not add any C14 analytical standard**. Your internal standard defines the analyte quantitation retention times. On your 3 separate runs, your internal standard retention time should not vary by more than a couple of seconds and your peak areas should not vary by more than 15%.
If you can't meet these requirements you equipment or method is broken. Likely problems include: "The large peak that you **think** is your Internal standard isn't" (grease from your fingers. plasticiser, detergents, general lab contamination, dirt in the system and/or contamination in the carrier gas?). Or, (less likely): leaks in the injector; or your column pushed too far into the injector.
Assuming that everything is OK now, you can make up the equivalent of a 100µM/L C14Acetate standard (25.6ng/µL) containing 23ng/µL of your internal standard. Now run this sample 3 times. The retention time window for your internal standard should still be within a couple of seconds. Your integration software will then use this to accurately lock onto your analyte. Your replicates for your analyte calculated from the internal standard should be better than 3%.
Now that you have got this to work, the dynamic range of your system for C15Acetate should go from 0.1µM/L (0.026ng on column) up to the maximum capacity of the system for C14Acetate which will be a maximum of <~120ng on column (or roughly 400µM). Sorry, you REALLY can't go above this - But what you can do is dilute your high concentration standard roughly 6 times with pure heptane. Your chromatography will still work. The software should still be able to find your internal standard at 23/6 = ~4ng (assuming that you do not have any other peaks within 2-4 seconds of the internal standard!). The clever software will do the calculations for you, as it is a simple linear relationship and you will get the correct result.  The bad news is that if you do inject a high concentration C14Acetate sample, the results will be complete rubbish because you have exceeded the design capacity of the system, BUT you can dilute those samples 6:1 with pure heptane and run them again. If the sample contains a number of other peaks near the internal standard and the instrument cannot find the internal standard,, dilute your sample about 1:6 with your internal standard solution and apply  a concentration correction factor.
So in conclusion the maximum dynamic range of your column with a FID is 0.02ng to 100ng injected onto the column. If you want to increase this at the top end, use a larger diameter/thicker film column like, say a 0.32mm ID column with a 1µm film thickness - The maximum sample capacity will be ~500ng on column (or roughly 2000µM/L of C14Acetate).
There is another alternative. Your bottom concentration is 15µM/L so you can cover the dynamic range that you want by running your standards diluted - You know that the top amount that you can inject is <100ng and the bottom detection limit of the system will be ~~0.02ng or roughly a 5000:1 dynamic range. So the have the top end dilution at 100µM/L, and go down down to <0.1µM/L but still keep the internal standard at 23 ng/µL (100µM/L).
Good Luck and Best Regards, Tim
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We have performed a set of injections using water and MeOH (in different lines) having a good S/N ratio in ESI- mode (compared with using ACN). A week later the same injections were performed and S/N was horrible. The base line has increased and is basically conformed of 262 and 218 m/z. This contamination appears only if MeOH and water are both running through the system, if it's just one of them the ions disappear. We have tried  changing the methanol  & water phases and the contamination remains there. We have also tried removing the column and still the same. However if ACN is used instead of MeOH the problem is solved.
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Looks to me like these peaks might be the sodium adducts of polyethylene glycols, try adding 0.1% formic acid to your mobile phases to see if these peaks disappear. Did you use Parafilm on or near the solvents? This could be the source of the polyethylene glycols. Never use sealing tape like Parafilm on or near the reservoir bottles or solvent lines on any LC/MS system because this type of contamination is possible. If you need to cover the reservoirs use aluminum foil with the dull side facing the solvent.
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Some venders say it not safe to use Sormal phase solvent with MS while some scientist it is ok. any clarifications??
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Polar phases work much better. The above reasons given for NP are more or less correct, but flammability should never be an issue in a dry nitrogen atmosphere (common to ESI). Normal phase mobile phases will work just fine IF you adapt them to the type of ionization source and instrument you have. For example: we routinely add adducts or doping agents to the mobile phase (or via infusions) to increase ion formation. Esp common in Negative mode (we run lots of Alcohol/Hexane mobile phases w/o a problem).
In all cases, watch the ESI needle (which should be in a pure N atmosphere) for sparking (bad, as it can damage the needle and result in lots of noise). Super low flow rates are more likely to cause problems so one quick fix is to increase the flow rate to suppress it.  Adding something more polar or conductive to the mobile phase will also cure the problem too.
These forums really are not the best place to go into detail about this, but anyone properly trained in LC/MS should know how to run most NP methods on their instrument w/o any danger or problems. *BTW: I professionally teach LC/MS operation and MD and we routinely run NP methods on most manufacturer's instruments. However, there are millions of possible methods so this is not to say that they all will work. Chromatography and MS are bit more complicated than that.
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What analytical technique is used to get the composition of streams from continuous distillation column (especially in refineries)? Is it GC, any other chromatography or any other technique?
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Any sensibility analysis starts by the determination of the number of degrees of freedom. A continuous distillation column has two degrees of freedom when the pressure is fixed. These two variables can be manipulated to produce desired streams purity e.g. reflux and distillate flow rate. To determine the distillate flow rate influence using rigorous simulation, fixing the feed flow rate and composition, it is necessary to choose the values for the number of stages for the stripping and rectifying sections and for the reflux. If any of these values are smaller than its minimal values, there will not be solution and neither convergence. Infinite reflux and infinite number of stages are the simplifying hypotheses which have inspired the name of the  ∞/∞ analysis. This analysis was firstly proposed by Petlyuk and Avet’yan (1971), but without any significant impact on the scientific community. The feasibility of the stream molar compositions obtained by the mass balance is checked by the existence of a residue curve from distillate to bottom (hypothesis of infinite reflux). Attainable flow rates for each column must be inside the interval from xB = xF (B = F and D = 0) to xD = xF (D = F and B = 0) (Bekiaris et al., 1996). The ∞/∞ analysis allows an easy and fast checking of the interrelation of the system streams in a column sequence without any column design consideration. For more details you can go to the link:
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