Identification Test: Goldenrod

Goldenrod

Identification Test: Transfer 1 g of sample in 20 ml of Petroleum ether and sonicate 20 min to a suitable flask, filter the sample to remove the Petroleum ether. Allow it dry, add 1 ml diluted HCl and 50 ml Ethyl Acetate, then sonicate 30 min. Filter the sample to remove the solvent, allow the residue to dry, add 2 ml MeOH, the residue dissolve.

Apply separate 10-ul portions of this solution and of a standard solution of USP Chlorogenic Acid RS in MeOH containing 0.5 mg per ml to the starting line of a thin-layer chromatographic plate (See chromatography <621>) coating with 0.25-mm layer of chromatographic silica gel mixture. Allow the spots dry, and develop the chromatogram in an unsaturated chamber with a solvent system consisting of a mixture of Toluene-Ethyl Acetate-Formic Acid-Glacial Acetic Acid-H2O is 1/ 15/ 1/ 1/ 2) until the solvent front have moved about 3/4 of the length of the plate. Remove the plate from chamber, air-dry and view under 365 nm long-wavelength UV-Light: the Rf value, color of the principal spot obtained from the test solution corresponds to that obtained from the standard solution.

From China Pharmacopoeia 2005 Volume 1- P218 .

If need,
HPLC Analysis of the flavonoids in pharmaceutical preparations from Solidago canadensis
See the paper: J. Pharm. Biomed. Anal. 32 (2003) 1045-2-53.
Results: NLT 0.20 % Chlorogenic acid in goldenrod....

Done....Night--- 11:05 PM July-15-2008

Linear-Solvent Gradient Dlution in HPLC

The linear-solvent-strength (LSS) model for gradient elution is based on an approximation for isocratic retention in RP-LC as a function of solvent strength.

Bonus Question: When run out of mobile phase, what is the consequence for the HPLC system...
A: all the hydraulic pathway would dry out
B: the air come into the pump, hydraulic pathway
C: the air would come into Column
D: the air could come into flow cell.
or what else....
( Tip: High school physical knowledge: air-pump...or try to recall when onsite setup the HPLC system, what need to do b4 start the pump at the first time?
Cool, one coke for the right answer, Mountain Dew

Peak Purity Analysis by Chemstation (2)

Peak Purity Analysis by Chemstation (1)

A key questions in general for chromatography and in particular for drug discovery is whether a given peak of interest represents co-eluting components. By using an algorithm in the LC/MS ChemStation to determining the number of components in a chromatographic peak. ( Just calculate a numerical value to characterize the degree of dissimilarity of the peak spectra, a so-called similarity factor, based on the match of the peak spectra to one another.)Two Curves

(1) Similarity curve: The mathematical fundamentals used in the similarity curve calculations are those used for the purity factor, however they are displayed in another format. All spectra from a peak are compared with one or more spectra selected by the operator, an apex spectrum for example. The degree of match or spectral similarity is plotted over time during elution. An ideal profile of a pure peak is a flat line at 1000.

(2) A threshold curve: shows the effect of noise on a given similarity curve. The effect increases rapidly towards both ends of the peak. In essence, a threshold curve is a similarity curve with background noise contribution.

In Summary, the threshold curve, represented by the broken line, gives the range for which spectral impurity lies within the noise limit. Above this threshold, spectral impurity exceeds the spectral background noise and the similarity curve intersects the threshold curve indicating an impurity providing the reference and noise parameters have been sensibly chosen.

Heavy Metals Assay: Can Not Print in my Varian 240 FS Atomic Absorption spectrometers

Today, Co-worker came to me and complained that issue of "Can not Print" " Loss the raw data" in the Varian AAS 24o FS.

Yes, there are some bugs in the Varian SpectrAA software 5.1 Pro.

How to fix : Called the technical support several times, was asked to pay attention on the report setting when "edit sequence parameters"/ then go to "Reports", select the "Print Report" and save the raw data.

That maybe help eliminate that problem.
After that, the "raw date missing" and "failed to print" gone..

Cool...

Ligusticum chuanxiong Hort

Identification:

(1) Weigh 1 g of sample in 5ml of Petroleum ether ( 30-60 degree C) into 10 ml tube, shake the tube 2 or 3 times in 10 hrs. Let it still, pipet the upper clear solution 1 ml in to a evaporating dish. Allow the solvent to evaporate, add 1 ml MeOH, the residues dissolve.

(2) then add 2% 3,5-Dinitrobenzoic Acid (in MeOH) testing solution 2-3 drops, and 2 drops of Sodium Hydroxide Solution (saturated dissolves in Methanol), display Purple & red.

From China Pharmacopoeia 2005 - P28.

Mass Spectra of Ferulic Acid (CAS# 537-98-4)
C10H10O4
Source Temperature: 150 °C,
Sample Temperature: 140 °C,
DIRECT, 75 eV,
Mass of molecular ion: 194.

Peak Data:

    51.0       5.5
   77.0       7.0
  105.0       5.7
  133.0      13.6
  145.0       5.0
  161.0       7.4
  176.0       5.2
  177.0       8.6
  179.0      19.0
  193.0       6.1
  194.0     100.0
  195.0      11.1
Low down the flow rate and injection concentration.

Dandelion

Assay of Caffeic Acid in Dandelion

Dandelion (Herb Taraxaci)

Identification: TLC.

Column : C18, 250 x 4.6, 5 um
Mobile Phase A: 0.01 M NaH2PO4, pH 3.8-4.0.
B : MeOH. A/B is 70/30.
Flow Rate: 1.0 ml/min
Detector Wavelength: 323 nm, 30 degree C.

Standard Prep: 7.5 mg std in 50 ml MeOH, shake & dissolve well, pipet 2 ml to 1o ml volumetric flask, bring to volume with MeOH.
Sample Prep: Weigh 1 g in 50 ml, fill to volume with 5% HOAc in MeOH.

Results: HPLC assay of Caffeic acid NLT 0.020 %. Correct me if i am wrong, to my best knowledge, there is some connection between Caffeic acid and carcinogenicity.

Pesticides on the Cucumber/Strawberry/Carrot

Today, just like to do something to kill the time....

The Difficulty is How to isolate/remove the polar pigments (chlorophyll and carotinoids) /fatty acids, organic acids before LC-MS/ GC-MS?

Strongly recommend the combine of PSA and GCB (PSA= primary-secondary amine, GCB = graphitized carbon black)

Steps:

(1) Sample preparation and extraction

Sample: 10g of cucumber/strawberries were homogenized and placed in a 50mL PTFE centrifuge tube.

Solvent: 10mL of acetonitrile were added to homogenate Shake for 1 minute, until uniform

Salts: 4.0g MgSO4 (anhydrous powder or granular), 1.0g NaCl, 1.0g trisodium citrate dihydrate
0.5g disodium hydrogencitrate sesquihydrate

Salts were added and vigorously shaken for 1 minute. Sample was centrifuged and the supernatant removed for cleanup. Pesticides standards (200ng/mL) were spiked in at this point

(2) Sample extract cleanup ( Here, Do not recommend any brand to hurt "Fair Play Spirit" )

1mL of supernatant from the previous step was placed into several 2mL polypropylene centrifuge tubes, each containing one of the following adsorbent mixes:

• 500 mg PSA
• 250-300 mg GCB,
• 1.2 g MgSO4
• 4.0 mL Acetone/Toluene(3/1)
• Shake and Centrifuge

Samples were shaken with the adsorbents for 30 seconds (carbon for 2 minutes), then centrifuged to produce a clear supernatant for GC/MS analysis.

GC-MS:
Column: A 30m, 0.18mm ID, 0.14µm.

Sample: Custom pesticide mix 200µg/mL each pesticide,

Inj.: 1.0µL splitless (Hold 1 min.)

Inj. temp.: 250°C

Carrier gas: Helium

Flow rate: Constant linear velocity @ 40cm/sec

Oven temp.: 40°C (hold 1 min.) to 320°C @ 12°C/min.

Det: Aiglent 6890N-5973 MSD

Transfer line temp.: 300°C

Ionization: Electron ionization

Mode: Selected ion monitoring (SIM)

Results:
Should soak/wash the fruits before eating
email me if want the assay results (not public, not panic).

Electrospray Summary

1. Analyte type:

1.1 Preformed ions (acids and bases)
1.2 Polar neutrals
1.3 Multiply charged ions of biopolymers
1.4 NMT<100>

2. Typical flow rates: low down - 1.0 ml/min
3. Promote ionization:
3.1 Correct pH
3.2 Favorable HPLC solvent composition
3.3 Post-column addition of reagents

4. Soft ionization technique

5. Typical applications: Drugs, Sugars, Peptides, Proteins, Oligonucleotides

Here is another question: Why GC-MS have a NIST/EPA/NIH Mass Spectral Library 2005 or 2008.? that is "Standard" Mass Spectral for GC-MS, it help newbie chemist would master the elucidation skill of Mass Spectral in 3 weeks.

However, Why LC-MS do not have any LC-MS Mass Spectral Library?
Answer: give me one Coke, i will tell you..... or call the Agilent/Waters/Varian/AB/PE Technical Support, they would answer...

Tips: Look at above # 4: comparing the ionization technique.
for LC-MS: ESI, APCI, APPI ( they are Soft technique), the ionization energy@interface different, the mass fragment different, ---- so not a universal mass library. Or or call the Agilent/Waters/Varian/AB Technical Support, ask them why develop so many "New and Individual" Patent ( on their own instrumentation only) on LC-MS ionization technique...

For GC-MS: EI ( Hard ionization technique), the Ionization energy (from 5 to 241.5 eV) is stable, so the molecular fragment should be the same....that is the reason why NIST/EPA/NIH have Mass Library.

It is so simple: understand the basic ionization technique, should answer.

Maximizing High Flow ESI Sensitivity

Today, met a general question: how to improve the ESI Sensitivity? So I would like to answer this question from the preparation of sample.

(1) Select appropriate chromatography grade HPLC solvents
(2) Avoid exotic solvent mixes (MeOH, MeCN, Water, 0.1% formic work best for 98% of LC/MS applications)
(3) Avoid adding excessive modifiers (eg amm.acetate @ 10mM not 50mM)
Choose the right column chemistry (C-8 vs. C-18); change column chemistry before changing solvent mix or composition.
(4) 2.1mm column or lower, flow rates of 200-400 uL/min
(5) Peak widths for quantification not greater than 8-10 secs
(6) Dissolve sample in start mobile phase solvent (weakest solvent possible).

Here is a schematic drawing of the Finnigan MAT 900S electrospray ion source (kindly provided by EPA/Dr. Helmut Muenster.)