Is that a silicon substrate?

The ability to measure any species in solution depends on the concentration, your sampling method, and of course the intrinsic Raman scattering efficiency. We have found that sampling the solution volume with a long focal length (eg x10) objective is the best. This paper shows the concentration dependence of the Raman of solutions of gelatin. 

"Improved Protocols for Vibrational spectroscopic analysis of body fluids",

Franck Bonnier, François Petitjean, Matthew J. Baker, Hugh J. Byrne,

Journal of Biophotonics, 7, 167-179 (2014) 

Thanks for the suggestion. Yes we use a silicon substrate.  I did try using a 10x objective but to no use. 

The longer focal depth objectives are better for focusing THROUGH a transparent volume, rather than the short objectives which are used to focus ON a surface. If you try to focus into, for example, a cuvette of water (from the top), you will see that you get the best signal (e.g. at 3300cm-1) with a longer focal depth objective. Then start adding your solute until you can start to see protein peaks at ~1600cm-1.

I am using a 1mg/ml protein sample. I place a drop of that solution onto a polished silica substrate and trying to get a peak. Should I change anything other than the 10x objective? Thanks in advance

It is easier to measure solutions by focussing "through" the solutions.

Use a deep vessel such as a cuvette or even a 96 well plate, and focus into the solution with a x10. You can check it with water, by maximising the signal at 3300cm-1, then check your protein solution.

I did try with a 96 well plate. I am able to get a big bump at 3300 cm-1 with the protein solution. But no other significant peaks. Please suggest something. Thanks in advance

Ok - water should also have a peak at about 1640cm-1. You should see if you can see this. 

If you can't see the proteins at this concentration, then the next option is to try to concentrate them - the easiest is by controlled evaporation. The ultimate concentration is completely dried, but then it is best to go back to the x100 on the deposited surface.

I loaded the 96 well plate with a very concentrated (1gm/ml) solution of the protein . I am able to get the water peak around 3300cm-1. I also get a big bump around 400-800 cm-1. I also tried with making a coffee ring with the protein solution on an ITO coated glass slide in vain. We have a 77mW 633 nm laser fitted with a 10x and 50x objective. I have tried various laser power and integration times as long as 3 minutes.

I do get peaks for the polymer from the 96 well plate. So I can distinguish between that and the peaks from the protein solution.

Please suggest some ways. Thanks in advance

I really don't know what else to suggest - the ultimate concentration would be the powder of the raw protein - do you get signals from this?

I do not have the powder form. Can you suggest anyway to optimize the instrument?

Do you have a powder of any form of biological material? Amino acids?

Yes I do.

Amino acids like glycine, histidine, leucine etc

I think anything - you can try to get a signal from a powder and then start to dilute it down.

I do get very good signals for powdered amino acids. However when i tried with powdered albumin I was unable to get any peaks. Please suggest something....... Thanks in advance

Do you just get a very broad background? If so - check this - we show how you can eliminate the background in raw proteins

"In vitro analysis of immersed human tissues by Raman microspectroscopy",

F. Bonnier, A.Mehmood, P. Knief, A. Meade, W. Hornebeck, H. Lambkin, K. Flynn, V. McDonagh, C. Healy,T.C. Lee, F.M. Lyng, H.J. Byrne,

Journal of Raman Spectroscopy, 42, 888–896(2011)

Your paper was very insightful. Yes we do get a very broad background. I have tried the technique suggested in your paper. I dissolved the protein in water and allowed a drop to dry on an ITO coated glass slide. Then the "coffee ring" that was formed was analyzed for Raman spectra. I got a very broad background. When i play around with the laser power (low laser power long integration time and vice versa) i either get a large background or the detector gets saturated. I have tried a lot of different combinations of integration time and laser power. Please suggest something......

Try the technique of putting the powder in a KBR pellet

I have got some peaks but I am skeptical about them having any meaning. I would like you to take a look at my spectra and give your input. Please give me your email id so I can send the image for your perusal. Thanks in advance

[email protected]

Yes we do have the 785 nm laser. I will try with Aluminium foil and Quartz substrate and let you know. Thanks in advance

We just tried our sample with another instrument working at 633 nm using silicon substrate and 10x objective. We got a clean protein spectra with all the marker bands. Hence the problem is with our instrument I guess. We have informed our engineers and lets see what they come up with.... Thanks for all your valuable inputs

Meanwhile we were trying out some less complex chemical compounds with the same instrument. We were able to get good peaks for dry chemical. But as soon as we dissolve it in water, we are unable to get any peaks. Any help is much appreciated.....

The protein molecule may have some fluorescence properties and blurred the Raman signal. In this case you need to enhance your Raman instrument. SERS!