>> Alright, well, good morning again.

So, today what I'd like to talk about is two

of three really important concepts in mass spect.

So, last time we introduced mass spect,

we talked about how the technique worked.

We introduced one big idea, and the big idea was

that you had to have an ion.

In the mass spect, we got that ion from kicking

out an electron, and then we talked about CI

and other soft ionization techniques like Mol D

and electrospray ionization,

and we said the big idea there is you get an ion

by adding a proton or adding a sodium to the molecule.

So, the three sort of big concepts

in mass spect really aren't very hard, and in fact,

nothing is hard about mass spect except we think differently

than we've sort of gotten used to thinking as organic chemists.

So, the first concept is exact mass.

Ever since you were, well, taking high school chemistry,

you got used to the molecular weights of compounds

and calculating them based on the average mass,

in other words, carbon S, carbon 12, and carbon 13,

so you have an average mass of carbon.

But here, each ion is being separated individually.

In other words, you're separating each ion

from each ion, which means you're going isotopomer

from isotopomer, and so we need to get used to the idea

of thinking about what the masses are

of the individual isotopes, and we'll explore some

of the implications of that.

So, the second big idea which ties

into that is isotopic abundances;

the fact different elements have different abundances

of different isotopes.

Carbon, for example, has 1.1% of its carbons as C13,

and the rest of them is carbon 12.

Sulfur has a little bit of S34, in addition to S34,

and we'll explore the implications of this,

and then in the last lecture, we'll talk about fragmentation.

So, this will be on Wednesday's lecture.

We'll discuss fragmentation as it pertains to EI mass spect,

which is really a subject or a special topic in its right.

We are already in the CI

in the electrospray ionization last time, so an example

of fragmentation of a quasi molecular ion

of a proteinated species.

Remember the type of molecular ion you get

from soft ionization chemistry,

and that's essentially just protic chemistry.

What we saw was a reaction that involving a leaving group

of proteinated group of nitrogen.

Leaving, that's chemistry that you've really been used

to since your sophomore year, but when we talk

about fragmentation and EI mass effect, we'll be talking

about fragmentation of radical cat ions, and given the fact

that in soft ionization techniques,

you don't generally get a lot of fragmentation.

Fragmentation isn't super important there,

but in EI mass effect, where you have tons of fragmentation,

you often can't even see your molecular ion.

It's very important.

So, I've given a couple of resources here,

which you'll using in your homework assignments.

These are also linked to the webpage for the course,

so you don't have to type in all of this stuff into the computer.

You can just click on the links that I've provided,

and as you'd go through the homework, you'll get familiar

with using these tools and choosing proper settings.

What I'd like to do now and today, is to really talk

about the concepts behind these first two issues over there,

isotopic matches and isotopic abundances.

And I thought maybe a place to explore this,

since we were talking about mass spect techniques last time,

and we talked about a variety of techniques, EI mass spect,

ESI mass spect, CI mass spect, is to introduce a variant of any

of those techniques,

any of those ionization techniques that's often referred

to HR mass spect or high resolution mass spect.

In general, when you get a mass spectrum,

the mass that you get is good to within a few tenths

or a few hundreds of a mass units.

In other words, if you get a number

like in one particular exercise, I'll show you later on,

the homework, you have number 1239.2.

You might say, okay, that number 1239.2 is probably good

to within a few 10ths of a mass unit.

However, with specialized instrumentation and calibration,

you can do far better.

So, with better instrumentation and calibration,

you can often get masses to very high precision with mass

to charge ratios to, I'll say high precision,

typically may 5 millimass units or 5 parts per million

or better, and I'll show you what I mean by that,

and in the case of a technique called ion cyclotron residence

mass spectrometry, sometimes even one magnitude better

than that.

So for example, by five parts per million,

I mean if I had a mass of 300.0000, then the mass

of a small molecule may be like a steroid or something

like that, we could get that within five parts in a million;

in other words, within .0015

or what a mass spectrometrist would refer

to 1.5 mmu or millimass units.

And, in this particular case, you can start

to distinguish among different species that have nominally,

in other words, to unit mass, the same mass.

So, the one thing, and I mentioned before is

that your molecular weight is going to be dictated

by your isotopes present.

[ Pause ]

So, let's take a moment to talk about isotopes and their masses.

So, if I go, and I look at our periodic table over there,

and I looked at carbon, and I look under atomic weight,

I'll see that the atomic weight is 12.001115 depending

on how many digits.

I'm sorry .01115, depending on how many digits they give you.

However, carbon is a mix of carbon 12 and carbon 13.

It's 98.9% carbon 12, and 1.1% carbon 13.

The mass of carbon 12 is set by definition as 12.00000,

as many 0's as you wish to write.

The mass of carbon 13, mass of carbon 13, is 13.00336.

So, when you go and say I want to weigh out a moll of carbon

or I want to weight out a moll of carbon-containing compound,

what you really doing is taking this number,

which is the weighted average of these two numbers.

In other words, 98.9% of this number and 1.1% of that number.

But, as I said you're separating molecules isotopomer

from isotopomer, and so you're going to get a peak

that corresponds to the isotopomer that's all C12.

Other elements also have isotopes.

Hydrogen, for example, if you look, your atomic weight

in the periodic table is 1.00794,

and yet hydrogen is a mix of hydrogen and deuterium,

sometimes called heavy hydrogen.

It's mostly, mostly, mostly hydrogen, 98.984% H1,

and only 0.16% H2, and so when you're thinking

about mass spectrometry, you want to use the mass of H1

and the mass of H1 is 1.00783.

[ Pause ]

Other common elements that you encounter in organic compounds;

nitrogen, the atomic weight is 14.067,0067.

This 00 there.

And yet, nitrogen N is a mixture of N14 and N15.

It's 99.62% N14 and 0.038% N15, and so the atomic mass of N14,

the mass that you would use in thinking

about mass spect is 14.00307.

Oxygen. I guess I'm not writing out the elements.

I'll just write 0.

Again, I'll give you the mass that you're used;

the atomic weight, it's 15.9994, and yet oxygen is a mixture

of oxygen 16, oxygen 17, and oxygen 18.

Oxygen 16 predominants at 99.76%.

There's just a tiny smidgen of oxygen 17,

.04% and just a little bit of O18, .20%.

And, so the mass when you're thinking

about mass spect is 15.99491 for oxygen.

Alright, so we've gone through some common elements here.

Let's take a moment to explore the implications of this.

You can see why this is really valuable, why the concept

of exact mass is really valuable.

So, let's take two simple molecules.

We'll take propane, CH3, CH2, CH3,

and we'll take acid aldehyde; CH3, CH0.

So, they're a nominal molecular weight of 42, of 44, pardon me.

But, with a high resolution mass spectrometer,

you can tell these molecules apart and more.

So, let's take a look.

If I have C12, H8; if I have C12, 3 and H1 8.

In other words, the predominant isotopomer of propane,

then we will see that it's exact mass is equal to 3 x 12.0000,

as many zeros as I choose to write plus 8 x 1.00783,

and when I tally that up, I get 44.0626.

If we compare our propane to our acid aldehyde, C12,

the main isotopomer is going to be C12 2, H1 4, O16,

and the exact mass is equal to 2 x 12.0000, etc., plus 4.00783,

plus 15.99491 is equal to 44.0262.

So, the point is that there is a significant difference

at the 100th place in mass

between the two different molecules.

That difference is 36.4 mmu, which actually is a lot,

as far as high resolution mass spectrometry, and what's nice,

is you can do more than just distinguish two molecules

from each other.

For a small molecule, you can have a computer give you all the

molecular formulas that fit the exact mass.

[ Pause ]

So, for example if you go to John Greaves

and give him a small molecule that you've synthesized,

and you get a high resolution mass spect on it,

and you get a particular exact mass,

he can give you all the molecular formulas that fit

within 5 or 10 millimass units.

Or you can go to that website I mentioned and linked

on the home page for the course or on the materials

for the course, and you can go ahead

and say I have an exact mass of 44.0262,

and then you give it some parameters, and you say,

give me all of the compounds that fit

within five parts per million or within 10 parts per million;

you don't want to give it too wide a range.

You don't want to give half a molecular weight unit,

or you'll get many compounds,

and you say those compounds could have up to 10 carbons

and up to, and you'd pull down from the menu say 40 hydrogens,

and up to 5 oxygens, and up to 5 nitrogens, and you'd find out.

And for a molecule this small, you'd find this is the only fit.

Or you'd find maybe just a couple of fits, but then some

of them would have absurd formulas like 5 nitrogens,

1 carbon, and 1 hydrogen.

Alright. Thoughts or questions at this point.

>> If you're doing positive ions, do you have to worry

about adding the exact mass of sodium?

>> Ah, beautiful question.

Well, you're in ions.

So, the question was, if you're doing positive ions,

do you need to worry about adding the mass of sodium.

So, of course, EI mass spect also gives you positive ions,

but there, you don't add a sodium or a proton,

but in the case of electrospray, you probably do have a sodium

or a proton or a potassium on there, so absolutely,

then you need to think about adding the exact mass of sodium

or potassium or one more proton on there.

And if you have a multiply charged species,

say with two protons on there, so a mass to charge that's based

on being dicad ion, then you'd add in two protons

or two sodiums, or a sodium and one proton in.

Great question.

>> This is only if you're looking at the parent peak.

>> This is only if you're looking at the parent peak.

And with EI mass spect, sometimes,

John Greaves will come back and say this is for a fragment,

but it's a logical fragment of your molecule,

where as with soft ionization you usually can get it.

Now, typically in small molecule mass spectrometry,

the journals basically say above a thousand molecular weight,

don't even both to do high resolution mass spectrometry,

because for small molecules like 300,

you really will get unique formulas.