Image: 

Asemblon is here to help you get the reaction you're looking for in the lab. Dr. Daniel Graham has written the follow answers to some of the most commonly asked questions. If you have further questions, please contact us!

Frequently Asked Questions

SELF-ASSEMBLY

• What is self-assembly?
• Where can I find a review paper about SAMs?
  

THIOLS

• Do thiols really smell?
• What type of facility is recommended for using thiols?
• Are there any health risks of using thiols?
• How should I store thiols?
• How long will thiols store?

PREPARING SELF-ASSEMBLED MONOLAYERS

• How do I make a 1 mMol solution of a thiol?
• How fast will a monolayer form?
• How do I get a certain surface composition when using mixed monolayers?
• Can I re-use a thiol solution? If so, do I need to worry about concentration changes in the solution?
• Will thiols assemble onto surfaces other than gold?
• The thiol I am using is not dissolving in the solvent I have chosen. What can I do?
• It seems that thiol self-assembly is always done in Ethanol. Is this true or can SAMs be prepared in other solvents?
• Does thiol self-assembly have to be done in 200 proof ethanol?
• Is the thiol attachment to a surface affected by anything such as heat or electric fields?
• How can I remove a SAM from a gold surface?

OBTAINING SPECIFIC TYPES OF SURFACE CHEMISTRY USING THIOLS

• Does Asemblon build molecules to order?
• I need to make a surface that is hydrophobic. Which thiols can I use?
• I need to make a surface that is hydrophillic. Which thiols can I use?
• What head groups would be useful for doing further chemical modification on a SAM?

ANALYZING SELF-ASSEMBLED MONOLAYERS

• How can I tell if I really formed a monolayer on the surface?
• How can I determine the order of a SAM?
• Are all thiol monolayers well ordered and crystalline?

SELF-ASSEMBLY
What is self-assembly?
Self assembly is the spontaneous organization of atoms or molecules into new, more complex, ordered structures. Self-assembly is the main mechanism used by nature in creating and sustaining life. DNA forms a double helix, proteins fold into precise configurations, and lipids organize into a cell membrane through self-assembly processes. These processes are controlled by the drive to minimize the energy of the system. In nature everything tends to stay in its lowest energy state unless acted upon by an outside force. For many systems the lowest energy state is obtained by arranging the molecules in a precise, ordered manner. For example the main components in cell membranes are lipid molecules. These molecules typically have a hydrophilic head group and one or two hydrophobic tails. Since cells are filled and surrounded with water, the lipids arrange themselves to maximize contact of the hydrophilic heads with the water inside and outside of the cells. This is done by forming a bi-layer with the tails arranged towards the middle of the bi-layer and the head groups on the outside that is exposed to the water environment.

The power of self-assembly can be captured through specific molecular design for the direct engineering of new, complex surfaces for applications ranging from the creation of non-fouling surfaces to the spontaneous assembly of molecular-scale computing elements. One way of tapping into the world of self-assembly is through the use of Asemblon high purity alkanethiols. Alkanethiols assemble on noble metal surfaces. The forces involved in this assembly are the interaction energy of the thiol group with the metal surface and the drive towards energy minimization through the interaction of the methylene carbons in the thiol backbone.

Where can I find a review paper about SAMs?

• An early review of SAMs (alkanethiol, silane and others) was written by Abraham Ulman (Chem. Rev. 1996, 96, 1533-1554).
• A review of the mechanism and kinetics of monolayer formation was written by Daniel Schwartz (Annu. Rev. Phys. Chem. 2001. 52:107–37).
• Chaki and Vijayamohanan wrote a review of SAMs as biosensors (Biosensors & Bioelectronics 17 (2002) 1–12).
• A review of SAMs and their applications was written by Frank Schreiber (J. Phys.: Condens. Matter 16 (2004) R881–R900).
• A comprehensive review of SAMs as a form of nanotechnology was written by Love et. al. (Chem. Rev. 2005, 105, 1103-1169).

THIOLS
Do thiols really smell?
Yes, most thiols have a foul odor. The extent of this odor depends somewhat on the chain length of the thiol. Shorter thiols tend to have a stronger odor. Part of the potency of the thiol smell comes from the noses acute ability to detect thiols in the part per million range. As a note, the main chemical that makes skunks smell so bad is a thiol, 2-butene-1-thiol. The thiol smell is also used in natural gas to help detect gas leaks. In this case the thiol used is typically ethanethiol.

What type of facility is recommended for using thiols?
Due to their smell, thiols should always be handled in a hood. Human noses can detect some thiols in the part per million range so even a small amount of thiol can make a room stink. If available, a fully vented hood is recommended. Otherwise a ductless hood with carbon filter should be adequate, especially for thiols with chain lengths of 11 carbons or higher.

Are there any health risks of using thiols?
Most thiols are considered noxious because of their foul odor. Some thiols have a degree of toxicity. As with any laboratory chemical it is recommended that thiols be handled within a hood and that personnel using the chemicals use protective gloves and lab coats. An MSDS is available for all Asemblon products. Contact support@asemblon.com if you would like an MSDS sheet for a particular compound.

How should I store thiols?
Thiols should be stored between +2 and +8 °C. They do not need to be frozen. Thiols should be stored under inert gas (N2 or Ar). This will minimize exposure to oxygen which can cause oxidation of the thiol groups. 

How long can I store a thiol?
If stored properly in a refrigerator under inert gas most thiols should be stable indefinitely. We typically recommend to use thiol products within 2 years of receipt.

PREPARING SELF-ASSEMBLED MONOLAYERS
How do I make a 1 mMol solution of a thiol?
First you need to know the final volume of the solution that you need to have. Then you can use the formula below to determine the mass of thiol you need to add to the determined volume to get a 1 mMol solution (other concentrations can be obtained in the same way by replacing the 0.001 mol/liter with the desired final concentration value).

If the thiol is a liquid, you can convert the mass to a volume using the density of the thiol. If the density is not given, a typical density for a thiol is around 0.84 g/ml.

How fast will a monolayer form?

Though it is somewhat dependent on the solution concentration, the formation of an initial monolayer occurs within seconds to minutes. After the initial monolayer has formed, the layer still contains gauche defects and is not fully ordered. Over time (hours to days) the layer will anneal out the gauche defects and come to a state where most all chains are in an all trans configuration and the layer has reached a stable ordered state. Though the assembly time may depend on the application and desired surface structure, in most cases assembly for a few hours will produce a good monolayer. In order to insure the layer has reached a semi-equilibrium state it is recommended to assemble for 24 hours or longer.

Schematic representation of thiol assembly on gold.
A). A gold sample is placed in a thiol solution.
B). Within seconds thiols start to adsorb onto the surface forming a partial monolayer.
C). Over minutes to hours the monolayers becomes complete and ordered.

The rate of assembly is also dependent on the head group chemistry and whether you use a thiol or a disulfide. Overall, disulfides have slower assembly kinetics than thiols. This means that if you use a mixed solution of thiol and disulfide, the thiol will most likely out-compete the disulfide for the surface and you will end up with a higher percentage of the thiol on the surface than the disulfide.

Understanding the assembly kinetics of a given thiol or disulfide become most important when working to create mixed monolayers. (see "How do I get a certain surface composition when using mixed monolayers" below).

How do I get a certain surface composition when using mixed monolayers?
Conceptually creating mixed monolayers is as simple as mixing the desired thiols in the correct proportions, placing a substrate in the solution to assemble, and allowing it to sit for a given amount of time. However in practice, it is often not this simple. The reason for this is that thiols assemble at different rates depending on the chain length and head group. Some general observations regarding competition of thiols for a surface include:

-Longer chain length thiols tend to assemble preferentially over thiols with shorter chain lengths when assembled from mixtures.
-Simple straight chain alkane thiols tend to assemble preferentially over thiols with bulky head groups when assembled from mixtures.
-Non-polar head groups tend to assemble preferentially over polar-head groups for thiols with similar chain length when assembled from mixtures.
-Thiols tend to assemble preferentially over disulfides when assembled from mixtures.

Since predicting the surface composition based on the solution composition is not directly possible, in order to create a surface with a given composition it will be necessary to create a calibration curve of surface chemistries from various solution compositions. This can be done by creating monolayers assembled from solutions of various compositions ranging from 100% of the first thiol to 100% of the second thiol and then determining the resultant surface composition of the monolayers. Once a calibration curve is created it should be possible to create a surface with a given composition with reasonable accuracy. (See the section on "Surface characterization" for information about determining surface composition of monolayers).

Regardless of whether you make a calibration curve or not, it is always recommended to adequately characterize the surface of a mixed monolayer to determine the surface composition of the thiols used in an experiment. This is especially important when trying to correlate surface properties with another measured property since you must know the surface concentration to make a valid correlation.

Can I re-use a thiol solution? If so do I need to worry about concentration changes in the solution?
Reusing a thiol solution is more of a question of storage than of concentration. Ten mL of a 1 mM solution contains approximately 6x1018 molecules. It takes approximately 4.8x1014 molecules to coat a 1 cm x 1 cm gold surface (assuming a flat surface and an average thiol footprint of 21 square angstroms). This suggests that you could coat around 12000 1 cm x 1cm samples before completely depleting a 1 mM solution of thiols. So even after reusing a solution several times the effective concentration should not change significantly.

If you plan to re-use a thiol solution you need to make sure it is stored properly. Thiols and thiol solutions should be stored after backfilling with an inert gas (nitrogen or argon) in a refrigerator. The inert gas backfill is done to minimize exposure of the thiols to oxygen. Oxygen exposure can cause oxidation of the thiol (R-SH) to a sulfate (R-SO3). The presence of some sulfate will generally not cause a problem since thiols have a much stronger affinity for the gold surface, but if a majority of the thiols become oxidized it can interfere with SAM formation.

When dealing with surface coatings we always recommend being over cautious and making fresh thiol solutions for critical experiments. This is mainly to avoid the risk contaminating the thiol solution through multiple uses and handling. Even small amounts of contaminants that are transfered to the solution can cause problems.

Will thiols assemble onto surfaces other than gold?
Yes. Thiol monolayers have been reported in the literature to assemble on a wide variety of metal surfaces including copper, silver, platinum, palladium, nickel, iron, mercury, TiO2, ZnSe, stainless steel, indium tin oxide, GaAs, and CdSe quantum dots. In many cases special surface pretreatments must be made to assure successful assembly. This often includes treatments to remove or reduce the surface oxide layers. The structure and coverage can vary from substrate to substrate, but in many cases thiols will form similar layers on these materials as they will on gold.

The thiol I am using is not dissolving in the solvent I have chosen, what can I do?
Sometimes if a thiol is not fully soluble in a chosen solvent, it can be dissolved by heating and/or sonication of the solution. If heating and/or sonication does not work, it may be necessary to lower your solution concentration or to choose a different solvent.

Alkanethiols with a chain length up to 16 carbons should be soluble in ethanol at a 1 mM concentration. An alkanethiol with 18 carbons or more will most likely need to be heated and sonicated to go into solution at a 1 mM concentration.

It seems that thiol self-assembly is always done in Ethanol. Is this true or can SAMs be prepared in other solvents?
Ethanol is probably the most common solvent used for thiol self-assembly. This may be due to the wide variety of thiols that are soluble in ethanol. Many other solvents have been used for self-assembly of thiols including, methanol, toluene, DMF, THF, water, and buffer solution.

Does thiol self-assembly have to be done in 200 proof ethanol?
There are many examples of self-assembly of thiols from other solvents, but most assembly is done from pure ethanol. Pure ethanol is recommended to eliminate the presence of water and other impurities that could interfere with the assembly.

It is important to remember that not all ethanol is created equally. Even 200 proof ethanol can contain traces of metals such as copper. If the levels of copper are too high it will deposit on the substrate during the assembly and can interfere with monolayer formation. Even if a given batch of ethanol meets the manufacturers specifications for trace metals, if the copper levels are high enough it will start to be detected on the gold surface. This is due to the high affinity between gold and copper. It doesn't take many atoms of copper to form a partial monolayer on the surface and start causing problems. If copper adsorbs on the surface it can disrupt monolayer formation.

Unfortunately there is no easy way to determine if the ethanol you have purchased contains too much copper. However one way to check is to soak a sample in the ethanol you intend to use and use electron spectroscopy for chemical analysis (ESCA) to check for the presence of copper. Below is a protocol for this:

-Take 2 clean gold samples
-Keep 1 as a control
-Soak the other sample overnight in the ethanol you intend to use
-Blow the sample dry with nitrogen
-Obtain the surface composition with ESCA. If no copper is detected the ethanol should be good to use. If copper is detected you should seek another source for ethanol.

Is the thiol attachment to a surface affected by anything such as heat or electric fields?
Temperature programmed desorption experiments have shown that thiols can be removed from the surface by heating. These studies suggest that thiol desorption happens in 2 phases, one starting at around 110º C and another around 300º C.

Studies have shown that thiols can be removed from a surface when a negative potential is applied to the surface. For this the sample is placed in an electrolyte solution in water or ethanol at either basic or neutral pH. Once removed from the surface the thiols go into solution. If the potential is removed from the sample, the thiols will reassemble onto the surface.

How can I remove a SAM from a gold surface?
A thiol monolayer can be irreversibly removed from a surface by cleaning the substrate in piranha solution. Piranha Solution typically consists of a 30:70 v/v solution of 30% hydrogen peroxide (H2O2) and concentrated sulfuric acid (H2SO4). This is a highly reactive mixture and should be used with extreme caution, only by those who have been properly trained. Piranha solution will literally eat the monolayer coating away from the surface. A procedure for using Piranha solution can be found in the Asemblon catalog.

Another way to remove a thiol monolayer from the surface irreversibly is to heat the surface with a butane torch. This must only be done with substrates that can withstand the intense heat of a butane torch. A few passes of the torch over the sample will vaporize the SAM layer from the surface.  It should also be noted that this method only works with gold layers with a titanium tungsten adhesion layer.  Other adhesion layer metals will migrate to the surface and contaminate the gold if they are heated with the butane torch.

OBTAINING SPECIFIC TYPES OF SURFACE CHEMISTRY USING THIOLS
Does Asemblon build molecules to order?
Yes, we provide custom synthesis. Pricing for custom synthesis is based on the time and difficulty of the synthesis.

I need to make a surface that is hydrophobic, which thiols can I use?
Any of the standard methyl terminated straight chain alkanethiols will produce a hydrophobic surface. These thiols assemble into close packed layers leaving mainly the terminal methyl group exposed to the surface. Methyl terminated alkanethiols with chain lengths of 9 carbons or more typically have contact angles greater than 110 degrees when assembled for at least 24 hours.

Asemblon products to produce hydrophobic surfaces:

• 1-Nonanethiol 091000-001
• Nonyl disulfide 181008-001
• 1-Undecanethiol 111002-001
• Undecyl disulfide 221009-001
• 1-Dodecanethiol 121004-001
• Dodecyl disulfide 241010-001
• 1-Hexadecanethiol 161006-001
• Hexadecyl disulfide 321011-001

I need to make a surface that is hydrophillic, which thiols can I use?
Hydrophillic surfaces can be created using any of Asemblon's hydroxyl, carboxyl or PEG terminated alkanethiols. 

Asemblon products to produce hydrophillic surfaces:

• Hydroxyundecanethiol 111013-002
• Hydroxyundecyl disulfide 221018-002
• Hydroxyhexadecanethiol 161016-002
• Hydroxyhexadecyl disulfide 321020-002
• 11-Mercaptoundecanoic acid 111021-003
• Carboxy undecyl disulfide 221025-003
• 16-Mercaptohexadecanoic acid 161023-003
• Carboxy hexadecyl disulfide 321026-003
• PEG 3 (1-Mercapto-11-undecyl)tri(ethylene glycol) 171041-011
• PEG 4 (1-Mercapto-11-undecyl)tetra(ethylene glycol) 191042-011
• PEG 6 (1-Mercapto-11-undecyl)hexa(ethylene glycol) 231043-011

What head groups would be useful for doing further chemical modification on a SAM?
Any head group chemistry that is used in traditional chemical reactions can potentially be used for further chemical modification on a SAM.

Asemblon products that can be used to provide reactive groups on SAMs:

• Hydroxyundecanethiol 111013-002
• Hydroxyundecyl disulfide 221018-002
• Hydroxyhexadecanethiol 161016-002
• Hydroxyhexadecyl disulfide 321020-002
• 11-Mercaptoundecanoic acid 111021-003
• Carboxy undecyl disulfide 221025-003
• 16-Mercaptohexadecanoic acid 161023-003
• Carboxy hexadecyl disulfide 321026-003
• 11-Amino-1-undecanethiol, hydrochloride 111027-004
• 11-Bromo-1-undecanethiol 111032-006

It should be noted that surface reactions can often be different than their solution counterparts. Reactions on SAMs should be successful as long as the reaction conditions do not destroy the underlying monolayer and the functional groups are not sterically hindered. Steric hindrance may be alleviated by using a mixed monolayer with the reactive group placed on a longer chain thiol that spaces it above the underlying layer. Mixed monolayers may also be advantageous since they can be used to space out the reactive groups latterly and thereby reduce steric problems for further reactions or molecular immobilizations.

For a review of reactions on SAMs, refer to the article “Reactions on Monolayers: Organic Synthesis in Two Dimensions” by Sullivan and Huck. For further information about chemical reaction on SAMs see "Chemical Reactions on Self-Assembled Monolayers" on page 59 of our catalog.

ANALYZING SELF-ASSEMBLED MONOLAYERS
How can I tell if I really formed a monolayer on the surface?
With some thiols this is very easy to determine. For example assembling a methyl terminated alkanethiol onto a gold surface will cause the surface to become hydrophobic. This can easily be visualized by simply pulling the sample out of the ethanol solution. Once the monolayer has formed the surface will no longer wet with ethanol and the sample will come out dry from the solution.

Ultimately the best way to determine this is to do surface analysis of the monolayer. There are many surface analysis methods that can be applied to SAMs. None of these techniques can tell you everything about your layer, but they each provide useful information.

Some surface analysis methods applicable to SAMs and what they can be used for can be seen in the table below.

How can I determine the order of a SAM?
Typically the order of a monolayer is determined by infrared spectroscopy (IR). This has to be done with an IR instrument equipped with a very sensitive detector and an accessory that enables measuring signal from a thin monolayer on a reflective surface. The order of the monolayer is assessed by looking at the peak positions of the symmetric vs and asymmetric va CH₂ stretches of the alkyl chain backbone. For well ordered alkane chains these values are reported as 2919 cm^-1 and below and 2851 cm^-1 and below, respectively.

Are all thiol monolayers well ordered and crystalline?
No. The order and crystallinity of a SAM film can depend on the chain length, head group, assembly time and solvent used. Some general observations include:

-Straight chain methyl terminated alkanethiols with less than 8 carbons tend to be somewhat disordered.
-Straight chain methyl terminated alkanethiols with more than 8 carbons, assembled for long times (hours), tend to be well ordered.
-Thiols assembled from dilute solutions (~1 µmol) for short times (seconds to minutes) tend to be disordered.
-Thiols with bulky head groups tend to disrupt order within the monolayer.