Chromatographic Method Development - Science topic
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Questions related to Chromatographic Method Development
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?
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.
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?
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.
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...?
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:
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
In my HPLC method development, Active peak cumming negative in sample and normal in standard solution. Can anybody suggest the reason behind this?
Mobile phase : 1% Aceetic acid in water and Acetonitrile 52:48 % v/v.
Diluent : Methanol
Column: Zorbax Eclipse Pluse
We are looking for a reliable method or procedure to identify all biological compounds in Cell Free Supernatant (CFS) of lactic acid bacteria.
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?
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.
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.
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.
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.
MOBILE PHASE A:buffer NH4HCO3 10 mM ? PH 10 : acetonitrile 60%:35%
MOBILE PHASE B: ACETONITRILE
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
post time 10'
i am surching for impurties of 1-, pipéridine-4-carboxamide
I need to detect styrene monomer from different food sample
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?
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
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!
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.
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.
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.?
We would be interested in 50mm column diameter scale. We are looking at Waters, Agilent, Shimadzu. Any comments on robustness? Customer support? Bad experiences?
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?
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 ?
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 ?
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?
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？
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.
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)?
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.
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.
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.
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
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?
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.
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?
Begineer for LC-MS
How to elute protein bound strongly to the beads? Does using loose beads helps or I should optimize the buffer solutions etc?
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
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....?
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!
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.
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.
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?
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?
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.
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?
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!
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
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.
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.
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
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.
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
- 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
- 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.
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?
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?
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
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.
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.
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!
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.
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?