Weeks V and VI Attachment
Hello again Y’all!
Indeed six good weeks have flown by and it’s my blogging once again, just right before I knew it. Hope all is fine there and that all your lil’ test tubes are still returning every cheeky grin of yours.
Alright, all things proper, in the previous entry, a brief overview of the major project was posted. So you’d probably know by now what metformin is and some theory about HPLC. With that, in this entry, I‘d be putting up some stuffs involved in the method development stage of the project.
Preparation of an Extemporaneous Metformin Sample
These procedures here are practically the same as most other usual practices in the lab. The only difference is that the sample arrives in a tablet form instead of the usual powder form; thus an additional step is required here, followed by the usual.
Materials:
- 500mg metformin tablet
- DI water
- sonicator
- Mortar and pestle
- 100ml volumetric flask
- glass funnel
- glass stirring rod
- 5ml dropper
- filter paper (optional)***
1. Thoroughly pulverize the metformin tablet into powder with the mortar and pestle.*
2. Rinse the drug particles off the pestle into the mortar with DI water.
3. Dissolve the powder using the stirring rod.
4. Transfer the drug solution into the volumetric flask using the glass funnel.
5. Rinse any remaining drug particles from the mortar into the volumetric flask.
6. Top up the rest of the volume with DI water till the 100ml mark, using the dropper when nearing the end.
You might be able to see a faint white line on the bottle immediately to the left.
A filled volumetric flask.
7. Shake the volumetric flask vigorously to dissolve any remaining powder in the solution.
8. Place the volumetric flask onto a sonicator** to aid in the dissolving process and removal of bubbles in the solution sample.
9. Filter the solution (optional) ***.
* You might notice that there is no weighing of any samples here to be dissolved, unlike that in the preparation for the standard sample. Instead, the entire tablet is used for the making of the sample.
** A sonicator is a machine that causes sonication, whereby sound (ultrasound) energy is used to agitate a sample for various samples such as that of speeding up the dissolving of a solute and the removal of gases (bubbles) from the sample.
*** Filtering is necessary for the sample in this experiment it would be used for HPLC. Thus, it would be removal of any undissolved particles would be necessary to prevent interference with the outcome of the results.
In this experiment, this sample is usually used as an unknown sample, whereby it is used to compare against a calibration plot from the standard and determine if it is accurate. In later phases of the project, it might also be used to facilitate a typical sample that would be made by patients themselves in stability tests.
Preparation of a Metformin Standard Sample
This procedure here uses a commercially available metformin standard, for the preparation of a solution with a concentration of 500ppm*. The procedures as you might probably notice are slightly different from that for the preparation of an extemporaneous metformin sample in that weighing is involved.
Materials:
- Meformin standard
This is the one we have in the lab.
- DI water
- Electronic pan balance
- 50ml** volumetric flask
- watch glass
- glass funnel
- glass stirring rod
- 5ml dropper
- spatula
- filter paper (optional)
Method:
1. Weigh out 25mg of metformin standard on the watch glass using the electronic pan balance.
2. Transfer the sample into the 50ml volumetric flask.
3. Rinse off any remaining metformin standard particles on the watch glass into the volumetric flask with DI water using the glass funnel to help collect the solution.
4. Top up the sample in the volumetric flask to 50ml, using the dropper when nearing the end.
5. Shake the sample vigorously to dissolve to metformin standard.
6. Sonicate the newly formed solution sample.
7. Filter the sample (optional).
* ppm denotes “parts per million”. The actual definition of it is quite complex and thus a simpler way of understanding it in this case is by looking at some examples such as 1mg/ 1000ml = 1ppm, 500mg/1000ml = 500ppm, 250mg/500ml = 500ppm etc. Essentially, the number of milligrams over a thousand milliliters is the number of parts per million.
** A 50ml volumetric flask has been selected here as it provides a sufficient amount for the carrying out of various tests within a suitable period of time while reducing unnecessary wastage of the standard (the standard is very expensive!).
In this experiment, the prepared metformin standard sample is usually used to chart a calibration plot for the determining of any samples of unknown concentrations and also to aid in determining the suitability a particular set of conditions for the quantification of metformin. Its concentration would usually be diluted to lower concentrations. Some commonly used concentrations are 50ppm and 100ppm.
An example of how dilutions are done is in the case of 50ppm for example, whereby 1ml of the 500ppm standard sample is transferred into a 10ml volumetric flask, with the rest of the volume being topped up with the mobile phase or DI water. Thus, simple calculations would confirm that 500ppm/10(DF) = 50ppm.
Alright, that’s all for this entry. Thanks for taking the time to read it and hope you’ve benefited somewhat. All the best to the rest of your attachments once again!
Alexander Soo TG02
0608122H
posted by THE CODEC 5 @ 11:37 PM
9 Comments:
hihi friend,
here the questions i wanna ask you.
how exactly sonicator remove the bubble ? i thought if we agitate the sample it will produce more bubble ? how ultrasound produce by the machine ?
Thanks =)
TINGJIE
TG02
0608495H
Hi alex
You smashed the tablet into small particles and used a sonicator to facilitate dissolving of the tablets right? Why not increase the temperature too? I believe increasing the temperature would quicken the rate of which the tablet dissolves.
My second question is, what is extemporaneous that you mentioned?
Thank you
Ernest
TG01
02/08/08
HELLO ALEX :D
we'll soon switch out places in another month's time...
okay here's my qn, the first test you have mentioned about. "this sample is usually used as an unknown sample, whereby it is used to compare against a calibration plot from the standard and determine if it is accurate." so any kind of drugs may be tested to determine if it is accurate in what way??
And, what do u mean by "it might also be used to facilitate a typical sample that would be made by patients themselves in stability tests"? What sample that is made by the patients?
Thanks (:
-Yvonne
Helloo my dear Readers,
Thanks for reading and for your questions.
To TINGJIE:
QNS:
how exactly sonicator remove the bubble ? i thought if we agitate the sample it will produce more bubble ? how ultrasound produce by the machine ?
ANS:
Well, a sonicator works by gently shaking/ vibrating the sample enough to allow the bubbles(air) to surface. The vibration is so subtle you'd barely notice it. It isn't the violent swirling or splashing of the sample. So bubbles would definitely not be formed. That said, the sonicator probably exploits the known fact that air has is lighter than water . It thus disturbs the molecules enough to create an inertia and allow the lighter air to float up from between the water molecules to the and surface.
The second possible way is that slight heat is produced during the process(due to vibrations), causing the water to warm up a little and increase in volume. Warm water has a lesser capability than cold water to retain gas molecules. Thus, this inevitably helps with the removal of gases too.
Finally, the ultrasound is produced with piezoelectric transducers that convert electrical energy into sound.
To Ernest:
QNS:
You smashed the tablet into small particles and used a sonicator to facilitate dissolving of the tablets right? Why not increase the temperature too? I believe increasing the temperature would quicken the rate of which the tablet dissolves.
My second question is, what is extemporaneous that you mentioned?
ANS:
Yes, raising the temperature would increase the dissolving rate. However, it would be an unnecessary and time consuming procedure as cooling would also be required. Besides, the tablet dissolves just fine at room temperature, and the sonicator is used in order to dissolve only unfortunate particles that remains, which might not always be the case. Thus, in other words, it would be too much a hassle for a rather simple problem.
Also, an extemporaneous solution is one that is made from a tablet, a commercially availble metformin tablets in this case. This is unlike the making of a solution from the respective raw materials and is less tedious.
To Yvonne:
QNS:
the first test you have mentioned about. "this sample is usually used as an unknown sample, whereby it is used to compare against a calibration plot from the standard and determine if it is accurate." so any kind of drugs may be tested to determine if it is accurate in what way??
And, what do u mean by "it might also be used to facilitate a typical sample that would be made by patients themselves in stability tests"? What sample that is made by the patients?
Ans:
As the values in the calibration plot are specific to metformin only, only a metformin sample can be used to determine if the plot is accurate. If another drug was to be used however, a different set of values would be formed as the values are relative to the conditions and parameters used in the machine, and thus different for different compounds.
As for your second question, as this project aims to allow patients to make their own extemporaneous metformin solutions from existing commercially tablets, stability tests would have to be carried out to determine if such an approach is suitable. Thus,the sample would be the extemporaneous sample itself.
Alrights. Hope that answers all of your queries. Thanks again!
Alexander Soo TG02
0608122H
hello!
why is a sonicator used to mix the particles? cant the normal vortexing be used?
and why is filtering optional?
yuxuan
Hi alex.
juz a few questions
u mention that the extemporaneous metformin sample is an unknown sample rite?
unknown meaning it is of unknown concentration?
If so, how u use that to determine whether ur calibration plot from standard is accurate?
Xin Ni
TG02
Group 9
This comment has been removed by the author.
hi alex
how long do you need to place the volumetric flask into the sonicator?? is it a optional thing or you must place it in no matter what??
Thanks.
Justina
TG01
0605950E
Hello all,
Once again thanks for reading and your questions.
To Yuxuan:
QNS:
why is a sonicator used to mix the particles? cant the normal vortexing be used?
and why is filtering optional?
ANS:
A sonicator is used to dissove any undissolved particles, not mixing a solution. So yeah. Hope that answers your first question. But in case you're still seeing question marks, a sonicator is not used to mix a sample but merely to dissolve undissolved particles by perhaps vibrating them so that they'd break up and dissolve into the water. However, a vortex works in a different manner in that it is a swirling of the entire sample of which might not affect the particles directly but merely just take them on a little "tour" about the solution. As for your second question, filtering filters out any unfortunate stubborn particles that still remain despite all efforts to dissolve them. These particles could easily cause a false result to occur, and at worst, spoil the column in HPLC. However, it is optional if you are not using the sample for HPLC but for some other experiments whereby particles in your sample would bot affect your process/ equipement.
To Xinni:
QNS:
u mention that the extemporaneous metformin sample is an unknown sample rite?
unknown meaning it is of unknown concentration?
If so, how u use that to determine whether ur calibration plot from standard is accurate?
ANS:
Actually, it was mentioned that an extemporaneous solution is "used as", or or acts as, an unknown sample, not "is an unknown sample". So yeah. I believe there's some misinterpretation on that. But in case you still might not understand, the concentration of the extemporaneous solution is known(for eg: 100ppm). But it is run as an unknown sample, of which the obtained result would be used to be compared against the calibration plot. Thus, if the result for 100ppm in the calibration plot is the same as the result for the extemporaneous solution, the calibration plot is very likely ro be accurate.
To Justina:
QNS:
how long do you need to place the volumetric flask into the sonicator?? is it a optional thing or you must place it in no matter what??
ANS:
Well, that's a good question. There's no fixed duration as to how long you SHOULD leave the sample in the sonicator. However, to play safe, I'd usually leave it in there for 15 to 30 mins. Also, it might be an optional thing depending on what you'd be using the sample for. But for the project that I'm doing, it's better to sonicate the samples to remove bubbles and dissolve the particles proper as both are a major problem to the system and a cause for false results if they are present.
Alrighty. Hope I'd answered your doubts nice and well. Thanks again y'all!
Alexander TG02
0608122H
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